RESUMEN
Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.
Asunto(s)
Dependovirus , Vectores Genéticos , Transfección , Humanos , Dependovirus/genética , Dependovirus/aislamiento & purificación , Células HEK293 , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Transfección/métodos , Plásmidos/genética , Plásmidos/aislamiento & purificación , Polietileneimina/química , Reactores Biológicos , Cromatografía por Intercambio Iónico/métodos , Virión/genética , Virión/aislamiento & purificaciónRESUMEN
In the rapidly advancing realms of gene therapy and biotechnology, the efficient purification of viral vectors is pivotal for ensuring the safety and efficacy of gene therapies. This study focuses on optimizing membrane selection for viral vector purification by evaluating key properties, including porosity, thickness, pore structure, and hydrophilicity. Notably, we employed adeno-associated virus (AAV)-sized nanoparticles (20 nm), 200 nm particles, and bovine serum albumin (BSA) to model viral vector harvesting. Experimental data from constant pressure normal flow filtration (NFF) at 1 and 2 bar using four commercial flat sheet membranes revealed distinct fouling behaviors. Symmetric membranes predominantly showed internal and external pore blockage, while asymmetric membranes formed a cake layer on the surface. Hydrophilicity exhibited a positive correlation with recovery, demonstrating an enhanced recovery with increased hydrophilicity. Membranes with higher porosity and interpore connectivity showcased superior throughput, reduced operating time, and increased recovery. Asymmetric polyether sulfone (PES) membranes emerged as the optimal choice, achieving â¼100% recovery of AAV-sized particles, an â¼44% reduction in model cell debris (200 nm particles), an â¼35% decrease in BSA, and the fastest operating time of all membranes tested. This systematic investigation into fouling behaviors and membrane properties not only informs optimal conditions for viral vector recovery but also lays the groundwork for advancing membrane-based strategies in bioprocessing.
Asunto(s)
Filtración , Membranas Artificiales , Nanopartículas , Tamaño de la Partícula , Nanopartículas/química , Filtración/métodos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Vectores Genéticos/aislamiento & purificación , Ensayo de Materiales , Materiales Biocompatibles/química , Animales , Albúmina Sérica Bovina/química , Bovinos , Sulfonas/química , Polímeros/químicaRESUMEN
Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.
Asunto(s)
Cerámica , Dependovirus , Durapatita , Vectores Genéticos , Serogrupo , Dependovirus/genética , Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Vectores Genéticos/genética , Humanos , Cerámica/química , Durapatita/química , Cromatografía/métodos , Células HEK293RESUMEN
Direct reprogramming is an emerging research field where you can generate neurons from a somatic cell, such as a skin or glial cell by overexpressing neurogenic transcription factors. This technique allows fast generation of subtype-specific and functional neurons from both human and mouse cells. Despite the fact that neurons have been successfully generated both in vitro and in vivo, a more extensive analysis of the induced neurons including phenotypic functional identity or gradual maturity is still lacking. This is an important step for a further development of induced neurons towards cell therapy or disease modeling of neurological diseases. In this protocol, we describe a method for functional assessment of direct reprogrammed neuronal cells both in vitro and in vivo. Using a synapsin-driven reporter, our protocol allows for a direct identification of the reprogrammed neurons that permits functional assessment using patch-clamp electrophysiology. For in vitro reprogramming we further provide an optimized coating condition that allows a long-term maturation of human induced neurons in vitro.
Asunto(s)
Diferenciación Celular/genética , Reprogramación Celular , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Reprogramación Celular/genética , Técnicas de Reprogramación Celular , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Humanos , Lentivirus/genética , Ratones , Técnicas de Placa-Clamp , Factores de Transcripción/genética , Transducción GenéticaRESUMEN
Progress in recombinant AAV gene therapy product and process development has advanced our understanding of the basic biology of this critical delivery vector. The discovery of rAAV capsid post-translational modifications (PTMs) has spurred interest in the field for detailed rAAV-specific methods for vector lot characterization by mass spectrometry given the unique challenges presented by this viral macromolecular complex. Recent concerns regarding immunogenic responses to systemically administered rAAV at high doses has highlighted the need for investigators to catalog and track potentially immunogenic vector lot components including capsid PTMs and PTMs on host cell protein impurities. Here we present a simple step-by-step guide for academic rAAV laboratories and Chemistry, Manufacturing and Control (CMC) groups in industry to perform an in-house or outsourced bottom-up mass spectrometry workflow to characterize capsid PTMs and process impurities.
Asunto(s)
Proteínas de la Cápside/genética , Dependovirus/genética , Regulación Viral de la Expresión Génica , Ingeniería Genética , Vectores Genéticos/genética , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Proteínas de la Cápside/metabolismo , Cromatografía Liquida , Dependovirus/metabolismo , Vectores Genéticos/aislamiento & purificación , Vectores Genéticos/normas , Glicosilación , Humanos , Polisacáridos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.
Asunto(s)
Vectores Genéticos , Lentivirus/crecimiento & desarrollo , Cultivo de Virus , Animales , Reactores Biológicos , Técnicas de Cultivo de Célula , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Humanos , Lentivirus/genética , Lentivirus/aislamiento & purificación , Transducción Genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Alphavirus-based vectors present an efficient approach for antigen preparation applied for vaccine development. Semliki Forest virus, Sindbis virus, and Venezuelan equine encephalitis virus have been engineered for high-level expression of antigens targeting infectious diseases and tumors. Alphaviruses possess a large application range as vectors can be delivered as naked RNA replicons, recombinant viral particles, and layered DNA plasmids. Immunization studies in animal models have provided protection against challenges with lethal doses of pathogenic infectious agents and tumor cells. So far, a limited number of clinical trials have been conducted for alphavirus vectors in humans.
Asunto(s)
Alphavirus/fisiología , Antígenos/inmunología , Vectores Genéticos/genética , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Alphavirus/aislamiento & purificación , Animales , Antígenos/genética , Línea Celular , Clonación Molecular , Ingeniería Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/aislamiento & purificación , Humanos , Inmunización , Ratones , Plásmidos/genética , ARN Viral/genética , Replicón , Transfección/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/aislamiento & purificación , Vacunas de Partículas Similares a VirusRESUMEN
The development of recombinant adeno-associated virus (rAAV) gene therapies is becoming an increasing priority in the biotherapeutic landscape. One of the challenges associated with the production of rAAV is the formation of empty AAV particles that do not contain a therapeutic gene. The concerns about the impact of empty particles on clinical safety and rAAV-mediated gene expression have necessitated the development of purification processes to remove these species. The development of a robust and scalable purification process to separate empty and full AAV particles at large scale remains a challenge. In this study, a novel anion exchange chromatography process based on isocratic wash and elution steps to enrich full rAAV2 particles is presented. An operating design space is identified to ensure the robustness of the process. The isocratic chromatography provides several advantages over the traditional shallow linear gradient elution, including lower buffer consumption, smaller intermediate pool volumes, and more robust manufacturing.
Asunto(s)
Dependovirus , Vectores Genéticos , Virión , Aniones , Cromatografía por Intercambio Iónico , Dependovirus/genética , Dependovirus/aislamiento & purificación , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Recombinación Genética , Virión/genética , Virión/aislamiento & purificaciónRESUMEN
In recent years, there has been a strong interest in the development and production of gene therapy products, especially those utilizing adeno-associated virus (AAV) particles. This is evident with the growing number of clinical successes and agency approvals for AAV therapeutics. Due to this increased investment in this technology, a need exists for scalable commercial production methods to ensure adequate product supply as research in AAV shifts from bench-scale development to clinical production. The purpose of this review is to summarize current scalable purification techniques that can be employed during the commercial manufacturing of AAV as well as highlight certain development considerations, such as adventitious agent removal and process development using the principals of quality by design.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/aislamiento & purificación , Cultivo de Virus/métodos , Dependovirus/crecimiento & desarrollo , Terapia Genética , HumanosRESUMEN
This protocol describes a simple single-step column purification (SSCP) of rAAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation.
Asunto(s)
Cromatografía de Afinidad/métodos , Vectores Genéticos/aislamiento & purificación , Heparina/química , Parvovirinae/aislamiento & purificación , Cromatografía de Afinidad/instrumentación , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Dependovirus , Vectores Genéticos/genética , Células HEK293 , Humanos , Parvovirinae/genética , Reproducibilidad de los ResultadosRESUMEN
Adeno-associated virus (AAV) recombinants are currently the vector of choice for many gene therapy applications. As experimental therapies progress to clinical trials, the need to characterize recombinant adeno-associated viruses (rAAVs) accurately and reproducibly increases. Accurate determination of rAAV infectious titer is important for determining the activity of each vector lot and for ensuring lot-to-lot consistency. The following protocol developed in our laboratory uses a 96-well TCID50 format and quantitative polymerase chain reaction (qPCR) detection for the determination of rAAV infectious titer.
Asunto(s)
ADN Viral/genética , Dependovirus/genética , Vectores Genéticos/genética , Genoma Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Algoritmos , Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Células HeLa , Humanos , Recombinación Genética , Reproducibilidad de los ResultadosRESUMEN
This is a simple method for rapid preparation of recombinant adeno-associated virus (rAAV) stocks, which can be used for in vivo gene delivery. The purity of these vectors is considerably lower than that obtained by either CsCl gradient centrifugation or by combination of iodixanol gradient ultracentrifugation followed by column chromatography.
Asunto(s)
Centrifugación/métodos , Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Ácidos Triyodobenzoicos/química , Animales , Células Cultivadas , Cesio/química , Cloruros/química , Cromatografía/métodos , Dependovirus/genética , Vectores Genéticos/genética , Humanos , Recombinación Genética , Reproducibilidad de los ResultadosRESUMEN
This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.
Asunto(s)
Centrifugación/métodos , Cromatografía por Intercambio Iónico/métodos , Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Ácidos Triyodobenzoicos/química , Virión/genética , Aniones , Cromatografía por Intercambio Iónico/instrumentación , Dependovirus/genética , Vectores Genéticos/genética , Concentración de Iones de Hidrógeno , Recombinación Genética , Reproducibilidad de los Resultados , Temperatura , Empaquetamiento del Genoma ViralRESUMEN
One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the establishment of CAR T cells.
Asunto(s)
Ingeniería Genética , Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Lentivirus/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Citometría de Flujo , Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Terapia Genética , Vectores Genéticos/aislamiento & purificación , Humanos , Inmunoterapia Adoptiva , Transducción Genética , Transfección , Transgenes , Ultracentrifugación/métodosRESUMEN
Advances in the use of lentiviral vectors for gene therapy applications have created a need for large-scale manufacture of clinical-grade viral vectors for transfer of genetic materials. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome of dividing and nondividing cells, resulting in long-term expression of the transgene both in vitro and in vivo. In this chapter, we present a method to transfect human cells, creating an easy platform to produce lentiviral vectors for CAR-T cell application.
Asunto(s)
Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Inmunoterapia Adoptiva , Lentivirus/genética , Receptores Quiméricos de Antígenos/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Genes Reporteros , Vectores Genéticos/aislamiento & purificación , Humanos , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , TransgenesRESUMEN
Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications and with the approval of Glybera (Alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20:1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme (GBM), a fatal form of brain cancer, and in malignant melanoma. In fact, Imlygic® (T-VEC, Talimogene laherparepvec, formerly known as OncoVex GM-CSF), displayed efficacy in a recent Phase-III trial when compared to standard GM-CSF treatment alone (Andtbacka et al., J Clin Oncol 31:sLBA9008, 2013), and has since become the first FDA-approved viral gene therapy product used in standard patient care (October 2015) (Pol et al., Oncoimmunology 5:e1115641, 2016). Moreover, increased efficacy was observed when Imlygic® was combined with checkpoint inhibitory antibodies as a frontline therapy for malignant melanoma (Ribas et al., Cell 170:1109-1119.e1110, 2017; Dummer et al., Cancer Immunol Immunother 66:683-695, 2017). In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are completely replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.
Asunto(s)
Terapia Genética , Vectores Genéticos , Herpesvirus Humano 1 , Animales , Chlorocebus aethiops , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Vectores Genéticos/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crecimiento & desarrollo , Herpesvirus Humano 1/aislamiento & purificación , Humanos , Transgenes , Células VeroRESUMEN
With the rapid development of gene therapy, gene-based medicine with adenovirus as vectors has become a new method for disease treatment. However, there are still enormous challenges in the large-scale production of adenoviruses for clinical use. Recent reports show that ion-exchange chromatography (IEC) is an effective tool for the isolation and purification of adenovirus. However, during the separation and purification, host cell protein and DNA, as well as serum from the culture medium, can non-specifically occupy numerous binding sites of the chromatography packings, thereby reducing the binding between the adenovirus and packing media. We here report a novel method for highly efficient purification of adenoviruses by increasing the salt concentrations of the samples to be ultrafiltrated by tangential flow filtration, the diafiltration buffer, and the samples for IEC purification. This method could significantly remove a large amount of serum proteins and host cell proteins, increase the amount of sample loaded on the IEC column, and improve the binding of the adenovirus samples to the packing media. A purity of > 95% could be obtained after one chromatography operation, and the number of purification steps and the amount of used packing media were reduced. The method is simple, economical, and efficient, and has excellent applications.
Asunto(s)
Adenoviridae/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Reactores Biológicos , Proteínas Sanguíneas , Tampones (Química) , Cromatografía por Intercambio Iónico , Células HEK293 , Humanos , Cloruro de Magnesio/química , Cloruro de Sodio/química , UltrafiltraciónRESUMEN
Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and developed an ELISA-like method named "VIRELISA" (virus receptor-linked immunosorbent assay) by adopting fusion with a streptavidin-binding peptide (SBP). It was demonstrated that optimized VIRELISA assays exhibited satisfactory performance for the titering of AAV2. The linear range of AAV2 was 1 × 105 v.g. to 5 × 109 v.g., with an LOD (limit of detection) of 5 × 104 v.g. Testing of VIRELISA for the quantification of AAV1 was also successful. Our study indicated that a generic protocol for the quantification of different serotypes of AAVs was feasible, reliable and cost-efficient. The applications of VIRELISA will not only be of benefit to laboratory research due to its simplicity, but could also potentially be used for monitoring the circulation AAV loads both in clinical trials and in wild type infection of a given AAV serotype.
Asunto(s)
Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Proteínas Recombinantes/genética , Dependovirus/genética , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/genética , Humanos , Transducción GenéticaRESUMEN
Krabbe disease is an inherited neurodegenerative disease caused by mutations in the galactosylceramidase gene. In the infantile form, patients die before 3 years of age. Systemic adeno-associated virus serotype 9 (AAV9) gene therapy was recently shown to reverse the disease course in human patients in another lethal infantile neurodegenerative disease. To explore AAV9 therapy for Krabbe disease, we engineered a codon-optimized AAV9 galactosylceramidase vector. We further incorporated features to allow AAV9-derived galactosylceramidase to more efficiently cross the blood-brain barrier and be secreted from transduced cells. We tested the optimized vector by a single systemic injection in the twitcher mouse, an authentic Krabbe disease model. Untreated twitcher mice showed characteristic neuropathology and motion defects. They died prematurely with a median life span of 41 days. Intravenous injection in 2-day-old twitcher mice reduced central and peripheral neuropathology and significantly improved the gait pattern and body weight. Noticeably, the median life span was extended to 150 days. Intraperitoneal injection in 6- to 12-day-old twitcher mice also significantly improved the motor function, body weight, and median life span (to 104 days). Our results far exceed the ≤70 days median life span seen in all reported stand-alone systemic AAV therapies. Our study highlights the importance of vector engineering for Krabbe disease gene therapy. The engineered vector warrants further development.
Asunto(s)
Dependovirus/genética , Galactosilceramidasa/genética , Terapia Genética , Vectores Genéticos/genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Transgenes , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Galactosilceramidasa/metabolismo , Expresión Génica , Orden Génico , Técnicas de Transferencia de Gen , Ingeniería Genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/aislamiento & purificación , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/fisiopatología , Ratones , Fenotipo , Transducción Genética , Resultado del TratamientoRESUMEN
With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.