Functional Assessment of Direct Reprogrammed Neurons In Vitro and In Vivo.

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.

VectorMOD: Method for Bottom-Up Proteomic Characterization of rAAV Capsid Post-Translational Modifications and Vector Impurities.

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.

Lentiviral Vector Bioprocessing.

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.

Main Features of DNA-Based Vectors for Use in Lactic Acid Bacteria and Update Protocols.

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.

Advances in Lentivirus Purification.

Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell therapies since they can stably integrate the genome in dividing and nondividing cells. LV production and purification processes have evolved substantially over the last decades. However, the increasing demands for higher quantities with more restrictive purity requirements are stimulating the development of novel materials and strategies to supply the market with LV in a cost-effective manner. A detailed review of each downstream process unit operation is performed, limitations, strengths, and potential outcomes being covered. Currently, the majority of large-scale LV manufacturing processes are still based on adherent cell culture, although it is known that the industry is migrating fast to suspension cultures. Regarding the purification strategy, it consists of batch chromatography and membrane technology. Nevertheless, new solutions are being created to improve the current production schemes and expand its clinical use.

Alphavirus-Based Antigen Preparation.

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.

Separating Empty and Full Recombinant Adeno-Associated Virus Particles Using Isocratic Anion Exchange Chromatography.

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.

Optimized Transgene Delivery Using Third-Generation Lentiviruses.

The lentivirus system enables efficient genetic modification of both dividing and non-dividing cells and therefore is a useful tool for elucidating developmental processes and disease pathogenesis. The development of third-generation lentiviruses has resulted in improved biosafety, low immunogenicity, and substantial packaging capabilities. However, because third-generation lentiviruses require successful co-transfection with four plasmids, this typically means that lower titers are attained. This is problematic, as it is often desirable to produce purified lentiviruses with high titers (>1 × 108 TU/ml), especially for in vivo applications. The manufacturing process for lentiviruses involves several critical experimental factors that can influence titer, purity, and transduction efficiency. Here, we describe a straightforward, stepwise protocol for the reproducible manufacture of high-titer third-generation lentiviruses (1 × 108 to 1 × 109 TU/ml). This optimized protocol enhances transgene expression by use of Lipofectamine transfection and optimized serum replacement medium, a single ultracentrifugation step, use of a sucrose cushion, and addition of a histone deacetylation inhibitor. Furthermore, we provide alternate methods for titration analyses, including functional and genomic integration analyses, using common laboratory techniques such as FACS as well as genomic DNA extraction and qPCR. These optimized methods will be beneficial for investigating developmental processes and disease pathogenesis in vitro and in vivo. © 2020 The Authors. Basic Protocol 1: Lentivirus production Support Protocol: Lentivirus concentration Basic Protocol 2: Lentivirus titration Alternate Protocol 1: Determination of viral titration by FACS analysis Alternate Protocol 2: Determination of viral titration by genome integration analysis.

Production of high-titer transmission-defective RNA virus-based episomal vector using tangential flow filtration.

In recent years, viral vector based in vivo gene delivery strategies have achieved a significant success in the treatment of genetic diseases. RNA virus-based episomal vector lacking viral glycoprotein gene (ΔG-REVec) is a nontransmissive gene delivery system that enables long-term gene expression in a variety of cell types in vitro, yet in vivo gene delivery has not been successful due to the difficulty in producing high titer vector. The present study showed that tangential flow filtration (TFF) can be effectively employed to increase the titer of ΔG-REVec. Concentration and diafiltration of ΔG-REVec using TFF significantly increased its titer without loss of infectious activity. Importantly, intracranial administration of high titer vector enabled persistent transgene expression in rodent brain.

Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification.

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.

Purification of Recombinant Adeno-Associated Virus 2 (rAAV2) by Heparin Column Affinity Chromatography.

This protocol describes a simple single-step column purification (SSCP) of rAAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation.

Sensitive Determination of Infectious Titer of Recombinant Adeno-Associated Viruses (rAAVs) Using TCID50 End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR).

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.

In vivo biodistribution analysis of transmission competent and defective RNA virus-based episomal vector.

RNA virus-based episomal vector (REVec) is an emerging viral vector system that mediates long-term stable gene expression in variety of cell types in vitro. However, little is known about its tissue tropism and persistence of gene expression in vivo. Here, to evaluate the feasibility of REVec for in vivo gene delivery, we conducted biodistribution analysis of transmission competent REVec and transmission defective ΔG-REVec in Lewis rats. Following intracranial administration of REVec, transgene expression was detected in various tissues. In contrast, transgene expression was only observed in the brain after ΔG-REVec administration. Low levels of vector shedding in the feces and blood and of neutralizing antibody in the serum were detected after REVec injection. In the brain, microglia, astrocytes and neurons were susceptible to REVec-mediated transduction. However, the animals administered with REVec, but not with ΔG-REVec showed a significant decrease in body weight compared to mock treated animals. Additionally, CD8 T cell infiltration was observed in the brain of these animals. In summary, we demonstrated that REVec promotes long-term transgene expression in vivo without causing high vector shedding or neutralizing antibody production; however, suggests the need to attenuate vector associated pathogenicity in the future.

Purification of Recombinant Adeno-Associated Viruses (rAAVs) by Iodixanol Gradient Centrifugation.

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.

Enrichment of Fully Packaged Virions in Column-Purified Recombinant Adeno-Associated Virus (rAAV) Preparations by Iodixanol Gradient Centrifugation Followed by Anion-Exchange Column Chromatography.

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.

Engineering HSV-1 Vectors for Gene Therapy.

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.

Preparation of Herpes Simplex Virus Type 1 (HSV-1)-Based Amplicon Vectors.

Amplicon vectors, or amplicons, are defective, helper-dependent, herpes simplex virus type 1 (HSV-1)-based vectors. The main interest of amplicons as gene transfer tools stems from the fact that the genomes of these vectors do not carry protein-encoding viral sequences. Consequently, they are completely safe for the host and nontoxic for the infected cells. Moreover, the complete absence of virus genes provides a genomic space authorizing a very large payload, enough to accommodate foreign DNA sequences up to almost 150-kbp, the size of the HSV-1 genome. This transgene capacity can be used to deliver complete gene loci, including introns and exons, as well as long regulatory sequences conferring tissue-specific expression or stable maintenance of the transgene in proliferating cells. For many years the development of these vectors and their application in gene transfer experiments was hindered by the presence of contaminating toxic helper virus particles in the vector stocks. In recent years, however, two different methodologies have been developed that allow generating amplicon stocks either completely free of helper particles or only faintly contaminated with fully defective helper particles. This chapter describes these two methodologies.

Production of Lentivirus for the Establishment of CAR-T Cells.

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.

Optimized Production of Lentiviral Vectors for CAR-T Cell.

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.

A novel method to purify adenovirus based on increasing salt concentrations in buffer.

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.