An Enhanced Retroviral Vector for Efficient Genetic Manipulation and Selection in Mammalian Cells.

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies.

Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex.

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.

Gene Editing by Ferrying of CRISPR/Cas Ribonucleoprotein Complexes in Enveloped Virus-Derived Particles.

The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.

Generation of Murine Chimeric Antigen Receptor-Modified T Cells for In Vivo Studies in Syngeneic Tumor Models.

CAR-T cell therapy has emerged as a potent and effective tool in the immunotherapy of refractory cancers. However, challenges exist in their clinical application, necessitating extensive preclinical research to optimize their function. Various preclinical in vitro and in vivo models have been proposed for such purpose; among which immunocompetent mouse models serve as an invaluable tool in studying host immune interactions within a more realistic simulation of the tumor milieu. We hereby describe a standardized protocol for the generation of high-titer γ-retroviral vectors through transfection of the HEK293T packaging cell line. The virus-containing supernatant is further concentrated using an inhouse concentrator solution, titrated, and applied to mouse T cells purified via a convenient and rapid method by nylon-wool columns. Using the method presented here, we were able to achieve high titer γ-retrovirus and highly pure mouse T cells with desirable CAR transduction efficiency. The mouse CAR T cells produced through this protocol demonstrate favorable CAR expression and viability, thus making them suitable for further in vitro/in vivo assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Production of γ-retroviral vectors from retrovirus-backbone plasmids Basic Protocol 2: Concentration of γ-retrovirus-containing supernatants Basic Protocol 3: Titration of concentrated γ-retrovirus Basic Protocol 4: Isolation and activation of mouse T cells Basic Protocol 5: Transduction of activated mouse T cells, assessment of CAR expression, and expansion of CAR T cells for further in vitro/in vivo studies Support Protocol: Surface staining of cells for flow cytometric assessment of CAR expression.

Therapeutic activity of retroviral replicating vector-mediated gene therapy in combination with anti-PD-1 antibody in a murine pancreatic cancer model.

Toca 511, a tumor-selective retroviral replicating vector encoding the yeast cytosine deaminase (yCD) gene, exerts direct antitumor effects through intratumoral prodrug 5-fluorocytosine (5-FC) conversion to active drug 5-fluorouracil by yCD, and has demonstrated therapeutic efficacy in preclinical and clinical trials of various cancers. Toca 511/5-FC treatment may also induce antitumor immunity. Here, we first examined antitumor immune responses activated by Toca 511/5-FC treatment in an immunocompetent murine pancreatic cancer model. We then evaluated the therapeutic effects achieved in combination with anti-programmed cell death protein 1 antibody. In the bilateral subcutaneous tumor model, as compared with the control group, enhanced CD8+ T-cell-mediated cytotoxicity and increased T-cell infiltration in Toca 511-untransduced contralateral tumors were observed. Furthermore, the expression levels of T-cell co-inhibitory receptors on CD8+ T-cells increased during treatment. In the bilateral subcutaneous tumor model, combination therapy showed significantly stronger tumor growth inhibition than that achieved with either monotherapy. In an orthotopic tumor and peritoneal dissemination model, the combination therapy resulted in complete regression in both transduced orthotopic tumors and untransduced peritoneal dissemination. Thus, Toca 511/5-FC treatment induced a systemic antitumor immune response, and the combination therapy could be a promising clinical strategy for treating metastatic pancreatic cancer.

Development of TaqMan-based real-time PCR based on ψ gene for quantitative detection of CAR-T cells.

Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = -3.3841x + 41.402 (R2 = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/µL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.

TRIM5α: A Protean Architect of Viral Recognition and Innate Immunity.

The evolutionary pressures exerted by viral infections have led to the development of various cellular proteins with potent antiviral activities, some of which are known as antiviral restriction factors. TRIpartite Motif-containing protein 5 alpha (TRIM5α) is a well-studied restriction factor of retroviruses that exhibits virus- and host-species-specific functions in protecting against cross-primate transmission of specific lentiviruses. This specificity is achieved at the level of the host gene through positive selection predominantly within its C-terminal B30.2/PRYSPRY domain, which is responsible for the highly specific recognition of retroviral capsids. However, more recent work has challenged this paradigm, demonstrating TRIM5α as a restriction factor for retroelements as well as phylogenetically distinct viral families, acting similarly through the recognition of viral gene products via B30.2/PRYSPRY. This spectrum of antiviral activity raises questions regarding the genetic and structural plasticity of this protein as a mediator of the recognition of a potentially diverse array of viral molecular patterns. This review highlights the dynamic evolutionary footprint of the B30.2/PRYSPRY domain in response to retroviruses while exploring the guided 'specificity' conferred by the totality of TRIM5α's additional domains that may account for its recently identified promiscuity.

Koala retrovirus and neoplasia: correlation and underlying mechanisms.

The koala retrovirus, KoRV, is one of the few models for understanding the health consequences of retroviral colonization of the germline. Such colonization events transition exogenous infectious retroviruses to Mendelian traits or endogenous retroviruses (ERVs). KoRV is currently in a transitional state from exogenous retrovirus to ERV, which in koalas (Phascolarctos cinereus) has been associated with strongly elevated levels of neoplasia. In this review, we describe what is currently known about the associations and underlying mechanisms of KoRV-induced neoplasia.

Retroviral PBS-segment sequence and structure: Orchestrating early and late replication events.

An essential regulatory hub for retroviral replication events, the 5' untranslated region (UTR) encodes an ensemble of cis-acting replication elements that overlap in a logical manner to carry out divergent RNA activities in cells and in virions. The primer binding site (PBS) and primer activation sequence initiate the reverse transcription process in virions, yet overlap with structural elements that regulate expression of the complex viral proteome. PBS-segment also encompasses the attachment site for Integrase to cut and paste the 3' long terminal repeat into the host chromosome to form the provirus and purine residues necessary to execute the precise stoichiometry of genome-length transcripts and spliced viral RNAs. Recent genetic mapping, cofactor affinity experiments, NMR and SAXS have elucidated that the HIV-1 PBS-segment folds into a three-way junction structure. The three-way junction structure is recognized by the host's nuclear RNA helicase A/DHX9 (RHA). RHA tethers host trimethyl guanosine synthase 1 to the Rev/Rev responsive element (RRE)-containing RNAs for m7-guanosine Cap hyper methylation that bolsters virion infectivity significantly. The HIV-1 trimethylated (TMG) Cap licenses specialized translation of virion proteins under conditions that repress translation of the regulatory proteins. Clearly host-adaption and RNA shapeshifting comprise the fundamental basis for PBS-segment orchestrating both reverse transcription of virion RNA and the nuclear modification of m7G-Cap for biphasic translation of the complex viral proteome. These recent observations, which have exposed even greater complexity of retroviral RNA biology than previously established, are the impetus for this article. Basic research to fully comprehend the marriage of PBS-segment structures and host RNA binding proteins that carry out retroviral early and late replication events is likely to expose an immutable virus-specific therapeutic target to attenuate retrovirus proliferation.

From Entry to the Nucleus: How Retroviruses Commute.

Once inside host cells, retroviruses generate a double-stranded DNA copy of their RNA genomes via reverse transcription inside a viral core, and this viral DNA is subsequently integrated into the genome of the host cell. Before integration can occur, the core must cross the cell cortex, be transported through the cytoplasm, and enter the nucleus. Retroviruses have evolved different mechanisms to accomplish this journey. This review examines the various mechanisms retroviruses, especially HIV-1, have evolved to commute throughout the cell. Retroviruses cross the cell cortex while modulating actin dynamics and use microtubules as roads while connecting with microtubule-associated proteins and motors to reach the nucleus. Although a clearer picture exists for HIV-1 compared with other retroviruses, there is still much to learn about how retroviruses accomplish their commute.

Studying Retroviral Life Cycles Using Visible Viruses and Live Cell Imaging.

Viruses exploit key host cell factors to accomplish each individual stage of the viral replication cycle. To understand viral pathogenesis and speed the development of new antiviral strategies, high-resolution visualization of virus-host interactions is needed to define where and when these events occur within cells. Here, we review state-of-the-art live cell imaging techniques for tracking individual stages of viral life cycles, focusing predominantly on retroviruses and especially human immunodeficiency virus type 1, which is most extensively studied. We describe how visible viruses can be engineered for live cell imaging and how nonmodified viruses can, in some instances, be tracked and studied indirectly using cell biosensor systems. We summarize the ways in which live cell imaging has been used to dissect the retroviral life cycle. Finally, we discuss select challenges for the future including the need for better labeling strategies, increased resolution, and multivariate systems that will allow for the study of full viral replication cycles.

Product Enhanced Reverse Transcriptase for assessing replication competent virus in vectors retroviral vectors pseudotyped with GALV and VSV-G envelopes.

We evaluated the use of the Product Enhanced Reverse Transcriptase (PERT) assay as a means of detecting virus in retroviral vectors products pseudotyped with Gibbon Ape Leukemia Virus (GALV) and Vesicular Stomatitis Virus G (VSVG) envelopes. PERT provides greater standardization than the S+/L- assay which has been used extensively in virus detection. A challenge is that PERT will also detect residual retroviral vectors as vector particles contain reverse transcriptase. Vector products were cultured for 3 weeks on HEK293 cells to amplify any potential virus. In addition, vector supernatant and end-of-production cells were spiked with GALV to evaluate for inhibition by the test article. Results of PERT and the S+/L- assay were compared. PERT and S+/L- assays were both effective in detecting virus. Vector supernatants were negative at the end of 3 weeks of culture by PERT for both GAVL and VSVG pseudotyped vector. In contrast, end-of-production cells were positive by PERT due to persistent vector producing cells. A one-week culture of cell-free media obtained at the 3 weeks timepoint allowed distinction of virus-free test articles from those with virus. The PERT assay is suitable for detecting replication competent retrovirus in vector products pseudotyped with GALV and VSVG envelopes.

Interferon-Stimulated Genes that Target Retrovirus Translation.

The innate immune system, particularly the interferon (IFN) system, constitutes the initial line of defense against viral infections. IFN signaling induces the expression of interferon-stimulated genes (ISGs), and their products frequently restrict viral infection. Retroviruses like the human immunodeficiency viruses and the human T-lymphotropic viruses cause severe human diseases and are targeted by ISG-encoded proteins. Here, we discuss ISGs that inhibit the translation of retroviral mRNAs and thereby retrovirus propagation. The Schlafen proteins degrade cellular tRNAs and rRNAs needed for translation. Zinc Finger Antiviral Protein and RNA-activated protein kinase inhibit translation initiation factors, and Shiftless suppresses translation recoding essential for the expression of retroviral enzymes. We outline common mechanisms that underlie the antiviral activity of multifunctional ISGs and discuss potential antiretroviral therapeutic approaches based on the mode of action of these ISGs.

Genotoxicity Associated with Retroviral CAR Transduction of ATM-Deficient T Cells.

Somatic variants in DNA damage response genes such as ATM are widespread in hematologic malignancies. ATM protein is essential for double-strand DNA break repair. Germline ATM deficiencies underlie ataxia-telangiectasia (A-T), a disease manifested by radiosensitivity, immunodeficiency, and predisposition to lymphoid malignancies. Patients with A-T diagnosed with malignancies have poor tolerance to chemotherapy or radiation. In this study, we investigated chimeric antigen receptor (CAR) T cells using primary T cells from patients with A-T (ATM-/-), heterozygote donors (ATM+/-), and healthy donors. ATM-/- T cells proliferate and can be successfully transduced with CARs, though functional impairment of ATM-/- CAR T-cells was observed. Retroviral transduction of the CAR in ATM-/- T cells resulted in high rates of chromosomal lesions at CAR insertion sites, as confirmed by next-generation long-read sequencing. This work suggests that ATM is essential to preserve genome integrity of CAR T-cells during retroviral manufacturing, and its lack poses a risk of chromosomal translocations and potential leukemogenicity. Significance: CAR T-cells are clinically approved genetically modified cells, but the control of genome integrity remains largely uncharacterized. This study demonstrates that ATM deficiency marginally impairs CAR T-cell function and results in high rates of chromosomal aberrations after retroviral transduction, which may be of concern in patients with DNA repair deficiencies.

Chromatin Immunoprecipitation of Retroviral Genomes with Antibodies Recognizing Modified Histones and Specific Viral Proteins.

Mammalian cells have developed and optimized defense mechanisms to prevent or hamper viral infection. The early transcriptional silencing of incoming viral DNAs is one such antiviral strategy and seems to be of fundamental importance, since most cell types silence unintegrated retroviral DNAs. In this chapter, a method for chromatin immunoprecipitation of unintegrated DNA is described. This technique allows investigators to examine histone and co-factor interactions with unintegrated viral DNAs as well as to analyze histone modifications in general or in a kinetic fashion at various time points during viral infection.

[Construction and Optimization of CD19 Chimeric Antigen Receptor T Cells Derived from C57BL/6J Mice].

OBJECTIVE: To explore the stimulation conditions, optimal culture time and infection time of C57BL/6J mice CD3+ T cells in vitro, so as to improve the infection efficiency of CD19 chimeric antigen receptor T cells (mCD19 CAR-T). METHODS: Purified C57BL/6J mice CD3+ T cells were cultured in anti-CD3/CD28 coated, anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated, respectively. The cells were stimulated in above three conditions for 12 h and 24 h, following with 24 h, 48 h and 72 h incubation and then the number of cell clones was recorded. C57BL/6J mice CD3+ T cells were stimulated for 12 h, 24 h, and 36 h under the above three conditions, then interleukin (IL)-2 (100 U/ml) was added. The number of cell clones was recorded under microscope at 24 h, 48 h, and 72 h of culture. After 24 h of stimulation, CD3+ T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells, and the percentage of GFP+ CAR-T cells was detected by flow cytometry. RESULTS: The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23% under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice. The number of clones of C57BL/6J mice CD3+ T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h. After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2, the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2, and the infection efficiency of CAR-T cells in this group reached 17.63%±4.17%. CONCLUSION: The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+ T cells are different from those of BABL/c mice. T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.

A case of T-cell acute lymphoblastic leukemia in retroviral gene therapy for ADA-SCID.

Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors.

Brief Histories of Retroviral Integration Research and Associated International Conferences.

The field of retroviral integration research has a long history that started with the provirus hypothesis and subsequent discoveries of the retroviral reverse transcriptase and integrase enzymes. Because both enzymes are essential for retroviral replication, they became valued targets in the effort to discover effective compounds to inhibit HIV-1 replication. In 2007, the first integrase strand transfer inhibitor was licensed for clinical use, and subsequently approved second-generation integrase inhibitors are now commonly co-formulated with reverse transcriptase inhibitors to treat people living with HIV. International meetings specifically focused on integrase and retroviral integration research first convened in 1995, and this paper is part of the Viruses Special Issue on the 7th International Conference on Retroviral Integration, which was held in Boulder Colorado in the summer of 2023. Herein, we overview key historical developments in the field, especially as they pertain to the development of the strand transfer inhibitor drug class. Starting from the mid-1990s, research advancements are presented through the lens of the international conferences. Our overview highlights the impact that regularly scheduled, subject-specific international meetings can have on community-building and, as a result, on field-specific collaborations and scientific advancements.

Identification and characterization of recent retrovirus in Rhinolophus ferrumequinum bats.

An investigation into retrovirus was conducted in six species of bats (Myotis aurascens, Myotis petax, Myotis macrodactylus, Miniopterus fuliginosus, Rhinolophus ferrumequinum, and Pipistrellus abramus) inhabiting South Korea. Exogenous retroviruses (XRVs) were detected in the tissue samples of R. ferrumequinum individuals by PCR assay. Proviruses were identified in all tissue samples through viral quantification using a digital PCR assay per organ (lung, intestine, heart, brain, wing, kidney, and liver), with viral loads varying greatly between each organ. In phylogenetic analysis based on the whole genome, the Korean bat retroviruses and the R. ferrumequinum retrovirus (RfRV) strain formed a new clade distinct from the Gammaretrovirus clade. The phylogenetic results determined these viruses to be RfRV-like viruses. In the Simplot comparison, Korean RfRV-like viruses exhibited relatively strong fluctuated patterns in the latter part of the envelope gene area compared to other gene areas. Several point mutations within this region (6,878-7,774 bp) of these viruses were observed compared to the RfRV sequence. One Korean RfRV-like virus (named Y4b strain) was successfully recovered in the Raw 264.7 cell line, and virus particles replicated in the cells were confirmed by transmission electron microscopy. RfRVs (or RfRV-like viruses) have been spreading since their first discovery in 2012, and the Korean RfRV-like viruses were assumed to be XRVs that evolved from RfRV.IMPORTANCER. ferrumequinum retrovirus (RfRV)-like viruses were identified in greater horseshoe bats in South Korea. These RfRV-like viruses were considered exogenous retroviruses (XRVs) that emerged from RfRV. Varying amounts of provirus detected in different organs suggest ongoing viral activity, replication, and de novo integration in certain organs. Additionally, the successful recovery of the virus in the Raw 264.7 cell line provides strong evidence supporting their status as XRVs. These viruses have now been identified in South Korea and, more recently, in Kenya since RfRV was discovered in China in 2012, indicating that RfRVs (or RfRV-like viruses) have spread worldwide.

Receptor-recognition and antiviral mechanisms of retrovirus-derived human proteins.

Human syncytin-1 and suppressyn are cellular proteins of retroviral origin involved in cell-cell fusion events to establish the maternal-fetal interface in the placenta. In cell culture, they restrict infections from members of the largest interference group of vertebrate retroviruses, and are regarded as host immunity factors expressed during development. At the core of the syncytin-1 and suppressyn functions are poorly understood mechanisms to recognize a common cellular receptor, the membrane transporter ASCT2. Here, we present cryo-electron microscopy structures of human ASCT2 in complexes with the receptor-binding domains of syncytin-1 and suppressyn. Despite their evolutionary divergence, the two placental proteins occupy similar positions in ASCT2, and are stabilized by the formation of a hybrid ß-sheet or 'clamp' with the receptor. Structural predictions of the receptor-binding domains of extant retroviruses indicate overlapping binding interfaces and clamping sites with ASCT2, revealing a competition mechanism between the placental proteins and the retroviruses. Our work uncovers a common ASCT2 recognition mechanism by a large group of endogenous and disease-causing retroviruses, and provides high-resolution views on how placental human proteins exert morphological and immunological functions.