Tool For Gene Delivery Research Articles

Targeted delivery of therapeutic agents to the heart.

For therapeutic materials to be successfully delivered to the heart, several barriers need to be overcome, including the anatomical challenges of access, the mechanical force of the blood flow, the endothelial barrier, the cellular barrier and the immune response. Various vectors and delivery methods have been proposed to improve the cardiac-specific uptake of materials to modify gene expression. Viral and non-viral vectors are widely used to deliver genetic materials, but each has its respective advantages and shortcomings. Adeno-associated viruses have emerged as one of the best tools for heart-targeted gene delivery. In addition, extracellular vesicles, including exosomes, which are secreted by most cell types, have gained popularity for drug delivery to several organs, including the heart. Accumulating evidence suggests that extracellular vesicles can carry and transfer functional proteins and genetic materials into target cells and might be an attractive option for heart-targeted delivery. Extracellular vesicles or artificial carriers of non-viral and viral vectors can be bioengineered with immune-evasive and cardiotropic properties. In this Review, we discuss the latest strategies for targeting and delivering therapeutic materials to the heart and how the knowledge of different vectors and delivery methods could successfully translate cardiac gene therapy into the clinical setting.

A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting

Inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensing photoreceptors. The most promising therapeutic approach for IRDs is gene supplementation therapy using viral vectors, which requires the presence of viable photoreceptors at the time of intervention. At later disease stages, photoreceptors are lost and can no longer be rescued with this approach. For these patients, conferring light-sensing abilities to the remaining interneurons of the ON circuit (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment strategy. Such treatments, however, are hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on the effective isolation of these cells to facilitate tool development. Herein, we describe a method to selectively isolate ON bipolar cells via fluorescence-activated cell sorting (FACS), based on the expression of two intracellular markers. We show that the method is compatible with highly sensitive downstream analyses and suitable for the isolation of ON bipolar cells from healthy as well as degenerated mouse retinas. Moreover, we demonstrate that this approach works effectively using non-human primate (NHP) retinal tissue, thereby offering a reliable pipeline for universal screening strategies that do not require inter-species adaptations or transgenic animals.

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy

ABSTRACT As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

Layered Double Hydroxide as a Potent Non-viral Vector for Nucleic Acid Delivery Using Gene-Activated Scaffolds for Tissue Regeneration Applications.

Nonviral vectors offer a safe alternative to viral vectors for gene therapy applications, albeit typically exhibiting lower transfection efficiencies. As a result, there remains a significant need for the development of a nonviral delivery system with low cytotoxicity and high transfection efficacy as a tool for safe and transient gene delivery. This study assesses MgAl-NO3 layered double hydroxide (LDH) as a nonviral vector to deliver nucleic acids (pDNA, miRNA and siRNA) to mesenchymal stromal cells (MSCs) in 2D culture and using a 3D tissue engineering scaffold approach. Nanoparticles were formulated by complexing LDH with pDNA, microRNA (miRNA) mimics and inhibitors, and siRNA at varying mass ratios of LDH:nucleic acid. In 2D monolayer, pDNA delivery demonstrated significant cytotoxicity issues, and low cellular transfection was deemed to be a result of the poor physicochemical properties of the LDH–pDNA nanoparticles. However, the lower mass ratios required to successfully complex with miRNA and siRNA cargo allowed for efficient delivery to MSCs. Furthermore, incorporation of LDH–miRNA nanoparticles into collagen-nanohydroxyapatite scaffolds resulted in successful overexpression of miRNA in MSCs, demonstrating the development of an efficacious miRNA delivery platform for gene therapy applications in regenerative medicine.

Viral Vectors as Gene Therapy Agents for Treatment of Glioblastoma.

Simple SummaryGlioblastoma is the most malignant cancer of the brain and current therapeutic strategies are clearly inadequate. In addition to surgical intervention, conventional drugs and ratio-therapy, scientists are looking at approaches based on gene therapy with genetically modified viruses. In this review we give a snapshot of the current state of play in this field of research and the available information about the clinical trials. We make some suggestions as to what opportunities could be explored further and hope that this review will stimulate discussion and conception of new life saving strategies.In this review, we scrutinize the idea of using viral vectors either as cytotoxic agents or gene delivery tools for treatment of glioblastoma multiforme (GBM) in light of the experience that our laboratory has accumulated over ~20 years when using similar vectors in experimental neuroscience. We review molecular strategies and current clinical trials and argue that approaches which are based on targeting a specific biochemical pathway or a characteristic mutation are inherently prone to failure because of the high genomic instability and clonal selection characteristics of GBM. For the same reasons, attempts to develop a viral system which selectively transduces only GBM cells are also unlikely to be universally successful. One of the common gene therapy approaches is to use cytotoxic viruses which replicate and cause preferential lysis of the GBM cells. This strategy, in addition to its reliance on the specific biochemical makeup of the GBM cells, bears a risk of necrotic cell death accompanied by release of large quantities of pro-inflammatory molecules. On the other hand, engaging the immune system in the anti-GBM response seems to be a potential avenue to explore further. We suggest that a plausible strategy is to focus on viral vectors which efficiently transduce brain cells via a non-selective, ubiquitous mechanism and which target (ideally irreversibly) processes that are critical only for dividing tumor cells and are dispensable for quiescent brain cells.

Particle bombardment technology and its applications in plants.

Particle bombardment, or biolistics, has emerged as an excellent alternative approach for plant genetic transformation which circumvents the limitations of Agrobacterium-mediated genetic transformation. The method has no biological constraints and can transform a wide range of plant species. Besides, it has been the most efficient way to achieve organelle transformation (for both chloroplasts and mitochondria)so far. Along with the recent advances in genome editing technologies, conventional gene delivery tools are now being repurposed to deliver targeted gene editing reagents into the plants. One of the key advantages is thatthe particle bombardment allows DNA-free gene editing of the genome. It enablesthe direct delivery of proteins, RNAs, and RNPs into plants. Owing to the versatility and wide-range applicabilityof the particle bombardment, it will likely remain one of the major genetic transformation methods in the future. This article provides an overview of the current status of particle bombardment technology and its applications in the field of plant research and biotechnology.

Recent Progress in Iron Oxide Nanoparticles as Therapeutic Magnetic Agents for Cancer Treatment and Tissue Engineering.

The positive response of superparamagnetic iron oxide nanoparticles (SPIONs), in terms of biodegradability, circulation, elimination, toxicity, and manipulation of their structure/activity relationship, has enabled them to find their way into commercialization as an iron supplement, MRI contrast agents, MPI tracers, and hyperthermia and magneto-mechanical actuators. This Review focuses on the most current progress regarding the application of SPIONs as magnetic therapeutic agents for cancer treatment and tissue engineering. Because of their superior magnetic anisotropy, irreversibility of high- and low-field magnetization, and superparamagnetic ordering at corporal temperatures, they exhibit the unique ability to respond to theraputic doses (e.g., in magnetic hyperthermia and targeted drug delivery). This Review discusses the role of SPIONs to enhance chemotherapy and radiotherapy efficiency and specificity and how this enhancement could mitigate some side effects. SPIONs applied as tools for gene delivery, immunotherapy, and tissue engineering are also reviewed in the context of their potential to translational medicine. Lastly, some emerging issues concerning SPION toxicity are summarized and how they are being addressed to achieve success in clinical applications is discussed.

Unraveling the Genome-Wide Impact of Recombinant Baculovirus Infection in Mammalian Cells for Gene Delivery.

Baculovirus expression systems have been widely used to produce recombinant mammalian proteins owing to the lack of viral replication in vertebrates. Although several lines of evidence have demonstrated impacts of baculovirus infection in mammalian hosts, genome-wide effects have not been fully elucidated. Here, we provide comparative transcriptome profiles of baculovirus and host-immune response genes in recombinant baculovirus-infected mammalian and insect cells. Specifically, to decipher the impacts of baculovirus infection in mammalian cells, we conducted total RNA-seq on human 293TT cells and insect Sf9 cells infected with recombinant baculovirus. We found that baculovirus genes were rarely expressed under the control of baculoviral promoters in 293TT cells. Although some baculovirus early genes, such as PE38 and IE-01, showed limited expression in 293TT cells, baculoviral late genes were mostly silent. We also found modest induction of a small number of mammalian immune response genes associated with Toll-like receptors, cytokine signaling, and complement in baculovirus-infected 293TT cells. These comprehensive transcriptome data will contribute to improving recombinant baculovirus as tools for gene delivery, gene therapy, and vaccine development.

Identification of a novel cell-penetrating peptide derived from the capsid protein of chicken anemia virus and its application in gene delivery.

Cell membranes are a great obstacle for entrance of gene therapeutic agents. Cell-penetrating peptides (CPPs) have been proven as a promising gene delivery tool. However, the early TAT peptide derived from the HIV transcription activator protein has been proven that the sequence contains Furin protease cleaved motifs which limited the TAT application in delivery of exogenous active molecules. In the present study, through the bioinformatics and experimental approach, we have identified a novel CPP derived from the N terminus of VP1 protein of chicken anemia virus (CAV), designated as CVP1-N2, which is rich in arginine residues and contains α-helical structure. Then, the ability of CVP1-N2 cell penetrating was detected using confocal imaging and flow cytometry. FITC-labeled CVP1-N2 peptide could rapidly internalize into different types of live cells with dose dependence and without cytotoxic effects by MTT assay. Surprisingly, CVP1-N2 with a pattern of nuclear sub-location has shown the higher uptake efficiency than TAT. At 10, 1, and 0.1 μM, the mean relative internalization of CVP1-N2 was respectively 1.08-, 12-, and 75-fold higher than that of CVP1, as well as 1.6-, 56-, and 75-fold higher than that of TAT. Moreover, using endocytic inhibitors along with low-temperature stress validated that the CVP1-N2 internalization route is direct translocation pathway. Finally, the capacity of CVP1-N2 for delivery of gene into cells was determined, where it was able to carry red fluorescent protein (RFP) and apoptin genes into cells respectively and induce the apoptosis. All these data indicate that CVP1-N2 could be used as a novel gene delivery vehicle for gene therapy in the future. KEY POINTS: • 1CVP1-N2 was identified as a novel more efficient cell-penetrating peptide. • 2. CVP1-N2 localized to the nucleus through the direct transduction pathway. • 3. CVP1-N2 was able to deliver the apoptin gene into HCT116 cells and induce apoptosis.

Constant companion: clinical and developmental aspects of torque teno virus infections.

Torque teno virus (TTV) is a commensal human virus observed as a circular single-negative-strand DNA molecule in various tissues and biological samples, notably in blood serum and lymphocytes. TTV has no apparent clinical significance, although it might be very useful as a prospective tool for gene delivery or as an epidemiological marker. Human populations are ubiquitously infected with TTV; the prevalence may reach 100%. The majority of babies become spontaneously infected with TTV, so that by the end of the first year of life, the prevalence reaches 'adult' values. TTV positivity in healthy early infancy and the presence of TTV in umbilical cord blood samples have been reported. The mechanism of infection and the dynamics of TTV prevalence in infants with age remain understudied. Meanwhile, the potential diagnostic and prognostic value of TTV as a marker deserves special attention and study, along with the possibility, causes and consequences of placental transmission of TTV under normal or pathological conditions.

Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy.

Viruses have been repurposed into tools for gene delivery by transforming them into viral vectors. The most frequently used vectors are lentiviral vectors (LVs), derived from the human immune deficiency virus allowing efficient gene transfer in mammalian cells. They represent one of the safest and most efficient treatments for monogenic diseases affecting the hematopoietic system. LVs are modified with different viral envelopes (pseudotyping) to alter and improve their tropism for different primary cell types. The vesicular stomatitis virus glycoprotein (VSV-G) is commonly used for pseudotyping as it enhances gene transfer into multiple hematopoietic cell types. However, VSV-G pseudotyped LVs are not able to confer efficient transduction in quiescent blood cells, such as hematopoietic stem cells (HSC), B and T cells. To solve this problem, VSV-G can be exchanged for other heterologous viral envelopes glycoproteins, such as those from the Measles virus, Baboon endogenous retrovirus, Cocal virus, Nipah virus or Sendai virus. Here, we provide an overview of how these LV pseudotypes improved transduction efficiency of HSC, B, T and natural killer (NK) cells, underlined by multiple in vitro and in vivo studies demonstrating how pseudotyped LVs deliver therapeutic genes or gene editing tools to treat different genetic diseases and efficiently generate CAR T cells for cancer treatment.

Engineering Corynebacterium glutamicum with a comprehensive genomic library and phage-based vectors

The Gram-positive bacterium Corynebacterium glutamicum sustains the industrial production of chiral molecules such as L-amino acids. Through heterologous gene expression, C. glutamicum is becoming a sustainable source of small organic molecules and added-value chemicals. The current methods to implement heterologous genes in C. glutamicum rely on replicative vectors requiring lasting selection or chromosomal integration using homologous recombination. Here, we present a set of dedicated and transversal tools for genome editing and gene delivery into C. glutamicum. We generated a cosmid-based library suitable for efficient double allelic exchange, covering more than 94% of the chromosome with an average 5.1x coverage. We employed the library and an iterative marker excision system to generate the carotenoid-free C. glutamicumBT1-C31-Albino (BCA) host, featuring the attachment sites for actinophages ϕC31 and ϕBT1 for one-step chromosomal integration. As a proof-of-principle, we employed a ϕC31-based integration and a Cre system for the markerless expression of the type III polyketide synthase RppA, and a ϕBT1-based integration system for the expression of the phosphopantetheinylation-dependent non-ribosomal peptide synthetase BpsA in the C. glutamicum BCA host. The developed genomic library and microbial host, and the characterized molecular tools will contribute to the study of the physiology and the rise of C. glutamicum as a leading host for drug discovery.

Anterograde Neuronal Circuit Tracers Derived from Herpes Simplex Virus 1: Development, Application, and Perspectives.

Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.

The Roles of Nanoparticles in Stem Cell-Based Therapy for Cardiovascular Disease.

Cardiovascular disease (CVD) is currently one of the primary causes of mortality and morbidity worldwide. Nanoparticles (NPs) are playing increasingly important roles in regulating stem cell behavior because of their special features, including shape, size, aspect ratio, surface charge, and surface area. In terms of cardiac disease, NPs can facilitate gene delivery in stem cells, track the stem cells in vivo for long-term monitoring, and enhance retention after their transplantation. The advantages of applying NPs in peripheral vascular disease treatments include facilitating stem cell therapy, mimicking the extracellular matrix environment, and utilizing a safe non-viral gene delivery tool. However, the main limitation of NPs is toxicity, which is related to their size, shape, aspect ratio, and surface charge. Currently, there have been many animal models proving NPs’ potential in treating CVD, but no extensive applications of stem-cell therapy using NPs are available in clinical practice. In conclusion, NPs might have significant potential uses in clinical trials of CVD in the future, thereby meeting the changing needs of individual patients worldwide.

Gene delivery to neurons in the auditory brainstem of barn owls using standard recombinant adeno-associated virus vectors.

Recombinant adeno-associated virus (rAAV) vectors are a commonly used tool for gene delivery. There is a large choice of different serotypes whose transduction efficiency varies for different animal species. In this study, three rAAV vectors were tested for transduction efficiency in the auditory brainstem of adult barn owls (Tyto alba) which are not standard laboratory animals. Injections with rAAV serotypes 2/1 and 2/5 resulted in reliable expression in various nuclei of the auditory brainstem of barn owls. Both vectors showed evidence of being spread by axonal transport. However, only rAAV2/5 also showed expression in regions far distant from the injection site, suggesting long-range axonal transport in connections along the auditory pathway. In contrast, injections with rAAV2/9 resulted in no expression. Our results demonstrate for the first time that commercially available rAAV vectors can be used for reliable gene expression in the barn owl auditory brainstem and pave the way toward optogenetic manipulation of neural activity in this important animal species in neuroethology and auditory physiology.

Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography

Lentiviral vectors (LVs) are a popular gene delivery tool in cell and gene therapy and they are a primary tool for ex vivo transduction of T cells for expression of chimeric antigen receptor (CAR) in CAR-T cell therapies. Extensive process and product characterization are required in manufacturing virus-based gene vectors to better control batch-to-batch variability. However, it has been an ongoing challenge to make quantitative assessments of LV product because current analytical tools often are low throughput and lack robustness and standardization is still required. This paper presents a high-throughput and robust physico-chemical characterization method that directly assesses total LV particles. With simple sample preparation and fast elution time (6.24 min) of the LV peak in 440 mM NaCl (in 20 mM Tris-HCl [pH 7.5]), this ion exchange high-performance liquid chromatography (IEX-HPLC) method is ideal for routine in-process monitoring to facilitate the development of scalable and robust LV manufacturing processes. Furthermore, this HPLC method is suitable for the analysis of all in-process samples, from crude samples such as LV supernatants to final purified products. The linearity range of the standard curve is 3.13 × 108 to 1.0 × 1010 total particles/mL, and both the intra- and inter-assay variabilities are less than 5%.

Abstract MP165: Exosome-mediated Encapsulation Alters AAV Antigenicity and Infectivity: Implications for Gene Delivery in the Heart

Gene therapy is a promising approach for the treatment of cardiovascular disease. Current strategies for myocardial gene transfer include the use of adeno-associated virus (AAV) vectors. However, AAVs may not be ideal for gene therapy vectors owing to pre-existing AAV capsid immunity in the human population that may reduce transduction efficacy and hinder preclinical-to-clinical translation. Interestingly, recent studies suggest that exosome-mediated encapsulation may protect viruses from neutralizing antibodies (NAbs) against the capsid and promote viral infectivity. Here, we describe the ability of exosome-enveloped AAVs, i.e. exosomal AAVs (eAAVs), to evade NAbs and serve as a highly efficient gene delivery tool for cardiovascular therapeutics. We have developed a method to purifiy eAAVs from AAV-producing HEK-293T cells, and used electron/confocal microscopy, qPCR, immunoblotting, dynamic light scattering and interferometric imaging measurements to characterize eAAV morphology, contents and mechanism of action. We confirmed eAAVs represent vesicular fractions that exhibit common exosome phenotype, along with the presence of virus particles, and demonstrated that eAAV infectious entry potentially involves trafficking via endocytic compartments. Using flow cytometry, Langendorff perfusion system and bioluminescence imaging, we then evaluated efficiency of heart targeting for eAAV9/eAAV6 and standard AAV9/AAV6 encoding for mCherry or firefly luciferase in human cardiomyocytes in vitro and in mouse model in vivo . Regardless of the presence or absence of NAbs, we showed that eAAVs are more efficient in transduction in the same titer ranges as compared to standard AAVs. To test therapeutic efficacy, we intramyocardially injected eAAV9 or AAV9 vectors encoding for SERCA2a in NAb+ post-myocardial infarction mice and further evaluated cardiac function using echocardiography. Remarkably, eAAV9-SERCA2a outperformed standard AAVs significantly improving cardiac function in the presence of NAbs (%EF 55.14 ± 3.50 compared to 27.31 ± 1.63 at 6 weeks, respectively). In summary, delivery of AAVs protected by carrier exosomes (i.e. eAAVs) may retain the clinical benefits of AAVs while addressing one of its major challenges.

Exosomes Derived from Human Placental Mesenchymal Stromal Cells Carrying AntagomiR-4450 Alleviate Intervertebral Disc Degeneration Through Upregulation of ZNF121.

Exosomes derived from mesenchymal stromal cells (MSCs) have emerged as novel drug and gene delivery tools. Current study aimed to elucidate the potential therapeutic role of human placental MSC (hPLMSC)-derived exosomes carrying AntagomiR-4450 (EXO-AntagomiR-4450) in intervertebral disc degeneration (IDD) progression. Initially, the differentially expressed miRNAs related to IDD were identified by microarray analysis, which provided data predicting the interaction between microRNA-4450 (miR-4450) and zinc finger protein-121 (ZNF121) in IDD. Next, miR-4450 and ZNF121 were elevated or silenced to determine their effects on the damage of nucleus pulposus cells (NPCs) treated with tumor necrosis factor α (TNF-α). The therapeutic effects of EXO-AntagomiR-4450 on NPCs were verified both in vitro and in vivo (15-week-old C57BL/6 male mice); especially gait analysis and fluorescent molecular tomography were used in live mice with IDD. Our results revealed that miR-4450 was highly expressed, while ZNF121 was poorly expressed in IDD patients and NPCs treated with TNF-α. Furthermore, miR-4450 was identified to specifically target ZNF121. In addition, the inhibition of miR-4450 exerted an alleviatory effect on the inflammation, apoptosis, and damage of the NPCs by upregulating ZNF121 (all P < 0.05). Moreover, EXO-AntagomiR-4450 retarded damage of NPCs in vitro, alleviated IDD damage, and ameliorated gait abnormality in vivo (all P < 0.05). hPLMSC-derived exosomes could be a feasible nanovehicle to deliver inhibitory oligonucleotides like AntagomiR-4450 in IDD.

Ocular delivery of CRISPR/Cas genome editing components for treatment of eye diseases.

A variety of inherited or multifactorial ocular diseases call for novel treatment paradigms. The newly developed genome editing technology, CRISPR, has shown great promise in treating these diseases, but delivery of the CRISPR/Cas components to target ocular tissues and cells requires appropriate use of vectors and routes of administration to ensure safety, efficacy and specificity. Although adeno-associated viral (AAV) vectors are thus far the most commonly used tool for ocular gene delivery, sustained expression of CRISPR/Cas components may cause immune reactions and an increased risk of off-target editing. In this review, we summarize the ocular administration routes and discuss the advantages and disadvantages of viral and non-viral vectors for delivery of CRISPR/Cas components to the eye. We review the existing studies of CRISPR/Cas genome editing for ocular diseases and discuss the major challenges of the technology in ocular applications. We also discuss the most recently developed CRISPR tools such as base editing and prime editing which may be used for future ocular applications.

A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts.

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as double-stranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [−] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.