Retroviral Producer Research Articles

CD33-TIM3 Dual CAR T Cells: Enhancing Specificity While Maintaining Efficacy Against AML

Background: The myeloid associated antigen CD33 is overexpressed in over 88% of Acute myeloid leukemia (AML) patients, making it a suitable target antigen for CAR T cell therapy across different genetic subtypes. Despite the development of clinical trial data, there remains a concern about on-target off-leukemia toxicity. To enhance specificity, we generated dual targeting CAR T cells that target CD33 and/or T-cell immunoglobulin and mucin-domain containing-3 (TIM3). The latter is known to be expressed on LSCs, but not on healthy hematopoietic precursor or stem cells. Notably, TIM3 is recognized for its inhibitory immunomodulatory role further enhancing the suitability of this target antigen. The objective of this research was to explore various CD33 and TIM3 dual-targeting CAR T cells approaches that enhance AML specificity and maintain high anti-AML activity. Methods: The hybridoma technology was used to generate antibodies against TIM3 immunized mice. Antibodies were screened for TIM3 specificity via ELISA and FACS. The anti-TIM3 single-chain variable fragment (scFv) DNA sequence was sequenced from hybridoma cells. Furthermore, the binding ability to TIM3 antigen (PDB ID: 5F71) was evaluated using AlphaFold. The CD33 scFv was derived from Gemtuzumab ozogamicin (clone: hP67.6). ScFv sequences and co-stimulation domains (CD28 or 4-1BB) were cloned into a pMP71 vector. Retrovirus for transduction was produced using the 293Vec-GALV and RD114 retroviral production system. In vitro co-culture assays of CAR T cells and target cells were performed to study the efficacy of CAR T cells. The cytotoxicity against wild-type and TIM3 transduced AML cell lines (THP-1 and OCI-AML3) was assessed by multiparameter flow cytometry (MPFC). The secretion of effector cytokines (IFN-γ, TNF, IL-2) was analyzed via CBA assays. In addition, avidity between CAR T cells and target cells was determined by Z-Movie analyzer. The different CAR constructs were screened for on-target off-tumor toxicity in colony forming unit assays (CFU) using isolated CD34 + hematopoietic stem and progenitor cells (HSPCs) from healthy doners after 14 days. In order to compare CAR constructs regarding long-term efficacy in antigen restimulation assays, CAR T cells were co-cultured with irradiated TIM3 transduced OCI-AML3 every 4 days at an E: T ratio of 1:1 for 24 days. Moreover, CAR T-cell proliferation, checkpoint marker expression and T-cell subset differentiation were analyzed via MPFC. Results: All dual CAR T cells (compound, split, tandem, pooled) were generated with high and robust transduction efficacy (Figure 1A). TIM3-dependent fratricide was not observed during CAR T cell manufacturing. Compared to single antigen targeting CAR T cells, dual CAR T cells showed enhanced cytotoxicity against AML cell lines and primary AML cells (Figure 1B). Moreover, we also observed a strong increase in the secretion of proinflammatory cytokines (IFN-γ and IL-2) and higher avidity of dual CAR T cells. Notably, split CAR T cells demonstrated greater specificity in cocultures directed against mono vs. dual-target antigen expression cell lines. In accordance with these observations, split CAR T cells did not exert on-target off-leukemia toxicity against healthy HSC in CFU assays. In the antigen restimulation assay, we observed that compound CAR T cells exhibited diminished expanding capacity and heightened expression of exhaustion markers in comparison to the other CAR T constructs. Conclusion: We developed multiple CD33 and TIM3 dual CAR T cells using both “AND” and “OR” gating strategies in this study. Our findings revealed that dual CAR T cells provided higher avidity and cytotoxicity compared to single targeting CAR T cells against dual antigen expressing target cells in vitro. Importantly, only the split CAR T constructs demonstrated specific killing of CD33 +TIM3 + cell lines and primary AML cells while sparing HSPCs. Prospectively, compound, tandem, pooled CD33 TIM3 specific CAR T cells might be helpful in a bridge to transplant setting, whereas split CAR T cells might provide a higher safety profile, thereby allowing a transplant independent approach. To advance our concepts, we are currently conducting in vivo experiments in an NSG mouse model.

Surface modification of PVDF hollow fiber ultrafiltration membranes for biopharmaceutical products, virus, and bacterial phage removal technology

In this study, radicals were chemically formed at the hydroxyl (-OH) sites on the surface of polyvinylidene fluoride (PVDF) hollow fiber membranes, and anionic-hydrophilic and hydrophobic materials were grafted onto the surface through -OH radical reaction to generate anions (−28, −41 mV). These membranes were then used as biomembranes (BMs) and showed excellent properties. We also studied the effects of chemical surface modification and reduction in pore size (60–130 nm) according to molecular weight. After anionic modification, the electrostatic repulsion between the pore surface and the bacteria prevents adsorption when anionic bacteria pass through the pores. Particles do not pass through the asymmetric pores due to agglomeration, resulting in separation. The hydrophilic membrane had a high initial water flux, but the flux gradually decreased due to fouling. In contrast, the hydrophobic modified membrane had a low initial water flux that eventually stabilized due to excellent fouling resistance. The protein recovery ratio (PRR) was 98.8 % for hydrophilic particles and 99.2 % for hydrophobic molecules, showing a higher PRR than the pristine membrane (96.4 %). An evaluation of removal of the gram-negative bacteria Brevundimonas diminuta (Bd) showed that no bacteria in membrane-purified solutions, confirming 100 % removal efficiencies. The developed membrane can be used in biopharmaceutical production and removal of proteins, retro viruses, and bacteria.

O genetski izazvanim bolestima u romanu „Kralj Bola i skakavac” (Król Bólu i pasikonik) Jaceka Dukaja

The author discusses the historical changes in attitudes towards infectious diseases in the mythological, Christian-religious and scientific worldview before and after the discovery of the causes of these diseases in the context of the types of futuristic fiction. One narrative line of the novel by contemporary Polish writer Jacek Dukaj King of Pain and the Grasshopper (Król Bólu i pasikonik, 2010) is centred on to the production of retroviruses and carcinogenic agents by genetic engineering companies that cause epidemics and destroy wildlife in the southern hemisphere. The text points out the specifics of the author’s descriptions of the cause of the plague and discusses Dukaj’s speculative projections of futuristic fiction.

Imaging Inflammasome Activation in Microglia.

Inflammasomes are multiprotein complexes that play key roles in the host's innate immune response to insult. The assembly of an inflammatory complex is initiated with the oligomerization of the upstream inflammasome-forming sensor and then follows a well-orchestrated multi-step process leading to downstream effector functions that are critical in the innate immune response. The final assembly of these steps provides a detectable readout of inflammasome complex activation in the form of an apoptosis-associated speck-like protein containing a CARD (ASC) speck. Inflammasome activation-and the release of IL-1β and ASC specks from the microglia, the brain resident immune cell-have been implicated in various neurological and neurodegenerative disorders. Protocols exist for the generation of fluorescent inflammasome indicator peripheral macrophages. Building upon these protocols, we describe here a protocol that details the generation of fluorescent inflammasome indicator microglia cells using recombinant retroviruses to transduce murine BV-2 cells. In this protocol, the cells are established in a manner to allow for experimental control of the initial priming step of the inflammasome activation process. We then provide a series of steps for using these reporter cells within an inflammasome activation assay and use real-time imaging of ASC-speck formation as an indicator of inflammasome activation. In addition, we describe strategies for using these cells for examining the effects of a test substance on inflammasome activation. This protocol offers an effective approach conducive to screening for and examining modifications of microglia inflammasome activation due to exposure to chemicals or pharmacological agents. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Production of retroviruses to express inflammasome indicator Basic Protocol 2: Generation of inflammasome indicator BV-2 cells Basic Protocol 3: Priming and activation of BV-2-ASC-Cerulean cells for inflammasome activation assay Basic Protocol 4: Examining modifications to inflammasome activation by test substances Basic Protocol 5: Imaging and analysis of ASC speck formation.

Genetically Engineered Porcine Organs for Human Xenotransplantation.

Xenotransplantation holds a promising future for many patients, especially those with end-stage renal disease or uncontrollable serum glucose levels. Porcine organs are viewed as the perfect candidate for a source of xenografts. However, the recipient's immunity, incompatibility of biologic systems, and transfer of new pathogenic organisms are all obstacles to clinical xenotransplantation, in addition to the risk of zoonosis and xenoantigens. Genetic modification of pigs using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) resulted in the production of porcine endogenous retrovirus (PERV)-free offsprings with the consequent removal of many clinical complications post-transplantation. Such as minimizing both acute and chronic inflammation, in addition to suppressing rejection reactions, which may prolong graft survival. To build on these recent successes, it is important to look at the limits of genetic engineering and develop ways to advance the field of xenotransplantation and reverse xenotransplantation clinical applications forward. Still, significant problems remain with clinical human xenotransplantation; future work should focus on developing an ideal genetically engineered swine donor source that can improve long-term graft survival and suppress the immune system in a clinically useful way.

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

Absence of porcine endogenous retrovirus (PERV) production from pig lymphoma cell lines

Porcine endogenous retroviruses (PERVs) -A and -B are integrated in the genome of all pigs, whereas PERV-C is found in many, but not all pigs. Some immortalized pig cell lines, among them lymphoma cells, but also mitogen activated primary lymphocytes have been shown to release virus particles, which were able to infect human cells and some of them were recombinant PERV-A/C. Since retroviruses can induce lymphomas, two newly established pig lymphoma cell lines and an older one (L23) were analysed for PERV expression. All three lines harboured PERV-A, PERV-B and PERV-C proviruses, but PERV-A/C recombinants were found only in the genome of L23 cells. The expression at the RNA level was very low and no protein expression and particle release was observed, suggesting that PERVs were not involved in the pathogenesis of these lymphomas. However, all three cell lines were infected with the porcine lymphotropic herpesvirus-3 (PLHV-3), which may have been involved in lymphoma development.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Virucidal Activities of Zinc-Finger Antiviral Proteins and Zinc-Binding Domains for Virus Entry, DNA/RNA Replication and Spread

The novel EBV (Epstein-Barr virus)-induced ZNFEB including its intronless locus and human protein variants, controls virus entry and exit from cell cycling in activated lymphocytes. ZNF ZCCHC3 binds RNA and facilitates viral RNA that is critical for RLR-mediated innate immune response to RNA virus. ZAP (Zinc-Finger Antiviral Protein) inhibits entry, replication and spread of certain viruses and promotes viral RNA degradation. ZAP may regulate DNA and RNA virus replication that ZAP inhibits Retroviral RNA production and HIV-1(Human Immuno-Deficiency Virus Type 1) infection by promoting the degradation of specific viral mRNAs. Futhermore, ZAP could regulate RNA virus degradation of SARS-CoV's (SARS Corona Virus) and MERS-CoV's (MERS Corona Virus) RNA virus. Replication of SARS-CoV requires proteolytic processing of the replicase polyprotein by a PLpro (Papain-Like Protease) that zinc conjugate inhibits SARSCoV PLpro protease activity. Zinc conjugated complexes as SARS-CoV 3C-like protease inhibitors play important role for this Zn2+-centered coordination pattern that the zinc-coordinating inhibitor is tetrahedrally coordinated. ZBD (Zinc-Binding Domain) is essential for formation of the functional Junin virus envelope glycoprotein complex. Complex ZBD regulates replicative arterivirus helicase and controls mRNA decay helicase. Viral inhibitor p53 down-regulates SARS-CoV replications that p53 inhibits replication of infectious SARS-CoV as well as of replicons and HCoV-NL63 (Human Coronavirus NL63). ZAP-70 kinase regulates HIV cell-to-cell spread that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts. Enveloped viruses enter cells and initiate disease-causing cycles of replication that in all cases virus-cell fusion is executed by one or more viral surface glycoproteins denoted as the fusion protein. Virucidal activities of ZNF, ZAP and ZBD are recognised by which Zn2+ ions bind RNA and facilitates viral RNA that is critical for RLR (RIG-1 Like Receptor)-mediated innate immune response to RNA virus and highly diverse fusion proteins have converged on the same overall strategy to mediate a common pathway of membrane fusion, causing to lead enhancement of the anti-viral activity. Zinc ions become used as Zn-coordinated inhibitors for viral regulation of virucidal activities.

Establishment of an induced pluripotent cell line from Taiwan black silkie chick embryonic fibroblasts for replication-incompetent virus production

The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2nd generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.

Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells

The γ-retroviral vector is a gene delivery vehicle that is commonly used in gene therapy. Despite its efficacy, its strong enhancers contributed to malignant transformations in some hematopoietic stem cell (HSC) gene therapy trials. A safer version without viral enhancers (SIN) is available, but its production is cumbersome, as high titers can only be obtained in transient transfection. Our aim was to develop a system that could easily generate high-titer SIN vectors from stable producer cells. The use of the cytomegalovirus enhancer-promoter sequence to generate the full-length genomic RNA combined to sequences that decrease transcriptional readthrough (WPRE and strong polyadenylation sequences) led to 6 × 106 infectious units (IU)/mL of a SIN GFP vector in transient transfection. The incorporation of a blasticidin selection cassette to the retroviral plasmid allowed the generation of stable clones in the 293Vec packaging cells that release 2 × 107 IU/mL and 1.4 × 107 IU/mL of a SIN GFP and a SIN PIGA vector, respectively. A titer of 1.8 × 106 IU/mL was obtained with a SIN vector containing the long 8.9-kb COL7A1 cDNA. Thus, an efficient process was established for the generation of stable 293Vec-derived retrovirus producer cells that release high-titer SIN vectors.

Generation of T Cell Receptor Retrogenic Mice.

The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus-mediated stem cell-based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector-encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

Production of High-Titer Retrovirus and Lentivirus Vectors.

The most commonly used method for production of retrovirus and lentivirus vectors in research laboratories is of transient transfection of 293T cells with a lentivirus vector plasmid, packaging genome plasmid(s), and an envelope expression plasmid. High-titer stocks are obtained by concentration of vector supernatants by ultracentrifugation.

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells.

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Clues to finding correlates of risk/protection for HIV-1 vaccines

Based on outcomes of informative HIV-1 vaccine prevention trials and the literature, it seems protection against HIV-1 acquisition more likely pertains to innate rather than adaptive immunity mechanisms. The proposed innate mechanism appears to be launched by alternatively activated macrophages in response to viral vectors and might be enhanced by natural female hormones. It was also suggested this novel immune mechanism was not likely amenable to discovery using standard or traditional approaches and is unlikely to be present in non-human models. A plausible, candidate innate mechanism with these characteristics pertains to the induction and production of human endogenous retrovirus–K102 (HERV-K102) particles by viruses which occurs in and generates foamy macrophages. HERV-K102 has salient features of non-pathogenic foamy retroviruses and its activation is part of the human-specific HERV-K HML-2 protective response described in HIV-1 patients. HERV-K102 particle production may be a novel and phylogenetically more recent form of innate immunity remarkably involving a viral anti-viral response. Accumulating clinical, biological and phylogenetic evidence supports a role of the HERV-K102 virus along with HML-2 responses, in the antagonism of HIV-1 replication and/or in the prevention of HIV-1 acquisition. Thus, it will be important to test the hypothesis that HERV-K HML-2 activation, HERV-K102 particle production and antibodies and T cell responses to select HERV-K102 or HML-2 antigens might comprise correlates of protection in HIV-1 vaccine prevention trials. Other proposed work would be to expand existing studies in HIV-1 highly exposed seronegative cohorts (HESN) and elite controllers to further evaluate a role of HERV-K102 and HML-2 in protection against HIV-1 replication and acquisition. The results of such inquiries may have important ramifications for the HIV-1 cure in addition to vaccines.

Generation of Innate Immune Reporter Cells Using Retroviral Transduction.

Innate immune cells are notoriously difficult to transfect; however, retroviruses can be used to stably integrate genes of interest into the host genome of primary or immortalized immune cells resulting in the generation of reporter cells. Here, we provide a detailed protocol covering the production of retroviruses, retroviral infection of innate immune target cells (including isolation and differentiation of murine bone marrow cells to macrophages), and several methods for enrichment of positively transduced cells.

Retroviral Transduction of Quiescent Murine Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation, cell and gene therapy applications, and the development of leukemia models for research. Because the hematopoietic progeny carries the genetic information of HSCs and replenishes the blood and immune system, corrective gene transfer into HSCs provides an ideal therapeutic approach for many monogenic hematological diseases and a useful tool for studies of HSC function and blood formation in normal and malignant hematopoiesis. However, the efficiency of gene transfer into HSCs has been limited by several features of viral vectors, viral titer, methods of viral transduction, and the property of stem cell quiescence. In this chapter, we describe the production of retrovirus using murine stem cell virus (MSCV)-based retroviral vectors and purification and transduction of murine HSCs.

Clues to finding correlates of risk/protection for HIV-1 vaccines

Almost a decade later, we still do not understand why in the STEP trial (2008), males with pre-existing antibodies to the Ad5 vector were associated with initial increased risk of HIV-1 acquisition. Similarly, we have little conclusive evidence of why in the RV144 trial (2009), vaccination with the ALVAC-HIV/AIDSVAX B/E was associated initially with almost a 60% vaccine efficacy at year one, which waned over 42 months to 31.2%, and where females were more protected than males. Based on the literature and trial outcomes, it was deduced that the elusive correlate of risk/protection may pertain to a novel, potent, innate protector mechanism launched by alternatively activated macrophages, which is probably induced by viruses and female steroid hormones. It was also suggested this mechanism was not likely amenable to discovery using standard or traditional approaches. A plausible, candidate mechanism was identified with these characteristics, namely the production of human endogenous retrovirus–K102 (HERV-K102) particles, which occurs in, and generates, foamy macrophages in vitro. Accumulating clinical, biological and phylogenetic evidence supports its role in the antagonism of HIV-1 replication and/or in the prevention of HIV-1 acquisition. Thus, it will be important to examine HERV-K102 particle production, increased integration and envelop antibody production as candidate correlates of protection in HIV-1 vaccine trials, as well as in HIV-1 highly exposed seronegative cohorts and elite controllers. The results of such efforts may have important ramifications for the HIV-1 cure in addition to vaccines.

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na+,K+-ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 107 infectious particles·ml-1). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.