Retroviral Gene Research Articles

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum-derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.

Structure of the Receptor Binding Domain of EnvP(b)1, an Endogenous Retroviral Envelope Protein Expressed in Human Tissues.

EnvP(b)1 is an endogenous retroviral envelope gene found in human and other primate genomes. We report EnvP(b)1 sequences in primate genomes consistent with an integration event between 40 and 71 million years ago. Using a highly specific polyclonal antiserum raised against the putative receptor binding domain (RBD) of human EnvP(b)1, we detected expression in human placenta, ovaries, and thymus. We found that EnvP(b)1 is proteolytically processed, and using cell-cell fusion assays in multiple primate cell lines, we demonstrated that extant EnvP(b)1 proteins from a variety of primate genomes are fusogenic. This work supports the idea that EnvP(b)1 is under purifying selection and its fusogenic activity has been maintained for over 40 million years. We determined the structure of the RBD of human EnvP(b)1, which defines structural similarities with extant leukemia viruses, despite little sequence conservation. This structure highlights a common scaffold from which novel receptor binding specificities likely evolved. The evolutionary plasticity of this domain may underlie the diversity of related Envs in circulating viruses.IMPORTANCE Organisms can access genetic and functional novelty by capturing viral elements within their genomes, where they can evolve to drive new cellular or organismal processes. We demonstrate that a retroviral envelope gene, EnvP(b)1, has been maintained and its fusion activity preserved for 40 to 71 million years. It is expressed as a protein in multiple healthy human tissues. We determined the structure of its inferred receptor binding domain and compared it with the same domain in modern viruses. We found a common conserved architecture that underlies the varied receptor binding activity of divergent Env genes. The modularity and versatility of this domain may underpin the evolutionary success of this clade of fusogens.

EPCO-36. GENOMIC INSTABILITY AND TRANSCRIPTOMIC SIGNATURES UNDERLYING EPIGENETIC MENINGIOMA SUBGROUPS REVEALS MECHANISMS OF IMMUNE INFILTRATION AND THERAPEUTIC VULNERABILITIES

Abstract BACKGROUND Meningioma treatments are limited due to incomplete understanding of meningioma biology. To address this, we performed multiplatform molecular profiling on 565 meningiomas with comprehensive clinical data to define genomic drivers and identify therapeutic vulnerabilities. METHODS DNA methylation profiling was performed on meningiomas from UCSF (n=200, discovery) and Hong Kong University (n=365, validation). Median follow-up was 5.6 years, and there were 388/142/35 WHO grade I/II/III meningiomas. Copy number variants (CNVs) were calculated for all meningiomas, and RNA sequencing was performed on UCSF meningiomas. Cell type deconvolution, metagenomics, CRISPR, and pharmacology were used for mechanistic and functional validation. RESULTS Unsupervised hierarchical clustering of differentially methylated DNA probes revealed that meningiomas were comprised of 3 epigenetic subgroups associated with good, intermediate, and poor outcomes, with representation from all WHO grades in each subgroup. Meningiomas from the subgroup with the best outcomes (52% WHO grade I) were distinguished by recurrent gain of Chr5. Meningiomas from the subgroup with intermediate outcomes (31% WHO grade II) were distinguished by genomic stability, enrichment of innate immune genes, and immune infiltration in the setting of endogenous retroviral gene re-expression, a mechanism of immune recruitment. The most aggressive subgroup of meningiomas (57% WHO grade III) was distinguished by genomic instability, including recurrent loss of Chr22q harboring NF2, and decreased immune infiltration. Consistently, NF2 suppression in primary meningioma cells derived from immunogenic meningiomas decreased expression of innate immune genes critical for immune recruitment, suggesting a novel immunostimulatory function of NF2. The most aggressive subgroup of meningiomas were further distinguished by activation of the mitogenic FOXM1 transcriptional program, and recurrent loss of Chr9p harboring CDKN2A/B, which rendered primary meningioma cells from this subgroup susceptible to CDK4/6 inhibitors. CONCLUSIONS Meningiomas are comprised of 3 epigenetic subgroups defined by genetic mechanisms driving immune infiltration in the tumor microenvironment and meningioma cell proliferation.

Multiplex CRISPR/Cas9 Genome Editing Targeting the BCL11A/HBG Axis Maximizes Fetal Hemoglobin Reinduction but Generates Chromosomal Translocations Which Persist In Vivo

Sickle cell disease and β-thalassemia are among the most common monogenic disorders globally, causing significant morbidity and early mortality. The only curative option available is allogeneic hematopoietic stem cell transplantation, which is limited by a lack of matched donors and risks, including graft versus host disease and secondary malignancy. Retroviral gene transfer is being explored in clinical trials, but an alternative approach is more targeted CRISPR/Cas9 genome editing, to recapitulate naturally occurring hereditary persistence of fetal hemoglobin (HPFH) which ameliorates disease. HbF reinduction is achieved by disrupting either transcription factor binding sites within the HBG promoter, or an erythroid enhancer sequence within the HbF repressor, BCL11A. Results from preclinical studies have suggested that HbF levels may remain suboptimal when each locus is targeted individually. We thus investigated the feasibility of a dual editing approach, targeting both loci simultaneously or sequentially, to maximize HbF production. G-CSF-mobilized human peripheral blood hematopoietic stem and progenitor cells (HSPCs) underwent CRISPR/Cas9 genome editing at either HBG promoter, BCL11A erythroid enhancer, or both loci targeted either simultaneously or sequentially. Immunodeficient mice were transplanted with edited cells. HSPCs cultured in differentiation media demonstrated comparable levels of editing at each locus in the single edited arms (by TIDE).The dual editing approach did not impair editing efficiency at each site when conducted sequentially and only slightly reduced efficiency in the simultaneously-edited reactions. HbF reinduction was also greatest in sequentially double-edited reactions, with HbF/HbA ratio as high as 3.9 times that seen in mock reactions by flow cytometry and 4.1 times by HPLC. In single-edited arms, HbF reinduction was slightly greater with HBG promotor than BCL11A erythroid enhancer editing. Two separate chromosomal translocation events encompassing both loci were detected in each double-edited arm and quantified using digital droplet PCR. Both were more frequent in simultaneous (mean of 1.0% and 0.6%) than sequential reactions (means <0.2%, n=3, p=0.0420 and p=0.0296). Analysis of single BFU-E colonies grown on methocult media revealed markedly different indel patterns at the 2 target loci. Concurrent hemoglobin fraction analysis of these clonal populations by HPLC demonstrated greatest HbF proportion in double-edited colonies. Where ≥60% editing was reported at both loci mean HbF was 71.3%, compared to 47.1% with ≥60% editing at HBG only, and 29.6% with ≥60% editing at BCL11A only (p=0.0373). Following transplant into mice, engraftment and lineage differentiation were comparable among all experimental arms. At necropsy, bone marrow populations of human CD45+ cells, CD34+CD38low HSPCs and, within these, HSC-enriched CD90+CD45RA- subpopulation, were present at comparable levels indicating that the dual editing approach did not impair engraftment. Translocation events were detected, albeit at frequencies of less than 0.25%, in the bone marrows of all 4 mice transplanted with simultaneously dual-edited cells and in 5 of 6 mice transplanted with sequentially dual-edited cells. Bone marrow cells cultured ex vivo demonstrated greatest HbF from mice transplanted with sequentially double-edited cells. In summary, we present evidence of maximized HbF reinduction with sequentially applied multiplex genome editing at BCL11A erythroid enhancer and HBG promoter, with no impairment of engraftment or differentiation. However, chromosomal translocation events were consistently seen in double-edited reactions, even when edits at each locus were applied sequentially, and persisted in vivo after engraftment, thus rendering this approach inappropriate for clinical applications. However, multiplex editing and targeting both the BCL11A erythroid enhancer and HBG promoter to maximize fetal hemoglobin induction may be a promising strategy for alternative platforms such as base editors which are expected to greatly reduce or completely eliminate the occurrence of translocations. Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Enochian: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees.

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.

VEGFR2 as a target for CAR T cell therapy of Ewing sarcoma

T cells engineered to express chimeric antigen receptors (CARs) are a novel modality to treat refractory cancers. The development of CAR T cells against Ewing sarcoma (EwS) is limited by a lack of targetable surface antigens. We investigated vascular endothelial growth factor receptor 2 (VEGFR2) expressed on tumor-associated blood vessels as potential CAR target in this cancer. Expression of VEGFR2 was studied by immunohistochemistry in human EwS biopsies and in murine xenografts and by flow cytometry in EwS cell lines. CARs with short, medium, and long hinge domains against either human or murine VEGFR2 were generated and expressed in human T cells by retroviral gene transfer. The capacity of the individual CARs to activate T cells in response to VEGFR2-expressing cells was compared in vitro. Tumor-associated endothelial cells in human EwS biopsies and in xenografts expressed VEGFR2. Tumor cells in the majority of EwS biopsies were also VEGFR2-positive. Following modification with anti-mouse or anti-human VEGFR2-specific CAR genes, T cells specifically lysed VEGFR2-expressing target cells of the respective species. CAR T cells with short-length or medium-length hinge domains were functionally superior over those with the long hinge region by in vitro parameters, including antigen-specific degranulation responses, lysis of tumor spheroids, tumor necrosis factor α secretion, sequential killing, and proliferation. VEGFR2 is consistently expressed on endothelial cells of the tumor stroma in EwS and thus is a candidate target for CAR T cells in this cancer. Among various VEGFR2-specific CARs, a construct with a short hinge domain was chosen to be further developed toward clinical translation.

Epidermal Growth Factor-Like Domain-8 (EGFL8) in Mammals: The Story so far


 
 
 Epidermal growth factor-like domain-8 (EGFL8) also known as vascular endothelial statin-2 (VE-statin-2). It was identified by using retroviral gene entrapment vectors, expressed in endothelial cells. It is located on chromosome6inhumansandchromosome17inthemouse. EGFL8codesaproteinof293aminoacidswithan amino-terminal signal peptide and has two EGF-like domains. EGFL8 plays a very important regulatory role in thymopoiesis, cell migration and invasion through the modulation of Notch signaling. Although the signaling regulatory factors of EGFL8 need to be explored but recent scientific advances have revealed some important aspect of its regulation. This review summarizes the current knowledge about all aspects of EGFL8 since its discovery.
 
 

Scaffold-Mediated Static Transduction of T Cells for CAR-T Cell Therapy.

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive clinical responses in patients with B-cell malignancies. Critical to the success of CAR-T cell therapies is the achievement of robust gene transfer into T cells mediated by viral vectors such as gamma-retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost-effective CAR-T cell therapies. Herein, a low-cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition is described. CAR-T cells produced by macroporous scaffold-mediated viral transduction exhibit >60% CAR expression, retain effector phenotype, expand to clinically relevant cell numbers, and eradicate CD19+ lymphoma in vivo. Efficient transduction is dependent on scaffold macroporosity. Taken together, the data show that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.

Optimization of canine CD20 chimeric antigen receptor T cell manufacturing and in vitro cytotoxic activity against B-cell lymphoma.

Canine B-cell lymphoma is one of the most common haematopoietic neoplasms in veterinary medicine, and it is considered a relevant model for human diffuse large B-cell lymphoma. Although the standard treatment consisting of multi-drug chemotherapy is effective in most cases, treatment is often challenging because of relapse and drug resistance. The adoptive transfer of autologous T cells genetically modified to express a CD19-specific chimeric antigen receptor (CD19 CAR-T cells) has been shown to be highly effective in human B-cell malignancies. However, there is no clinically available canine CAR-T cell therapy. We generated canine second-generation and third-generation CAR-T cells by retroviral gene transduction. Optimization was performed to investigate effective viral transduction protocols and favourable culture conditions for canine CAR-T cells. The RetroNectin-bound virus infection method resulted in more than 70% transduction efficiency. The effect of culture conditions on the phenotype of CAR-T cells was evaluated by the expression of surface markers. in vitro cytotoxicity assays of target cells cultured with CD20 CAR-transduced cells demonstrated that CD20 CAR-T cells exhibit cytotoxicity against CD20-expressing canine B-cell lymphoma cells and canine CD20-transduced murine cells, whereas no effect was observed against cells that lacked canine CD20 expression. Our study established virus-based canine CAR-T cell generation, providing fundamental data for a better understanding of canine adoptive T-cell therapy.

287-OR: TCR/HLA Humanized Mice Reveal Reduced Tolerance and Increased Immunogenicity of Posttranslationally Modified GAD65 Epitope

T cells specific for post-translationally modified (PTM) islet antigens have been implicated as critical players in the development of type 1 diabetes. T cells reactive to the deamidated version of glutamic acid decarboxylase 65 (GAD65) 115-127 epitope accumulate in peripheral blood of patients as they progress to disease. However, knowledge regarding the development, activation, differentiation and islet recruitment of PTM reactive T cells is limited. Using retroviral gene delivery, we have developed a humanized TCR/HLA mouse model to assess the in vivo function of human TCRs reactive to either the wild type (GADWT) or the deamidated (GAD120E) GAD65115-127 epitope. Both TCRs supported efficient thymic selection; however, fewer regulatory T cells developed in mice expressing the PTM reactive TCR, which was partially rescued by the thymic delivery of GAD120E peptide. Immunization of immune sufficient polyclonal DR4.NOD mice with GAD120E/CFA elicited a stronger response compared to GADWT/CFA, confirming that tolerance to the wild type autoantigen is more robust. Interestingly, while islet infiltration in GAD120E single TCR mice was dependent on priming with the cognate PTM peptide, GADWT-TCR T cells could be induced to infiltrate islets after priming with either GADWT or GAD120E peptide, despite the vastly different functional affinity (EC50, GADWT: 0.047μM vs. GAD120E: ≥1.4μM). These findings suggest that the development and expansion of GAD65 PTM reactive CD4+ T cells are less restricted in peripheral lymphoid organs, but their recruitment to the tissue site is dependent on the availability of PTM antigen. Disclosure Y. Jing: None. Y. Kong: None. J. McGinty: None. G. Blahnik-Fagan: None. M. Bettini: None. E.A. James: None. M. Bettini: None. Funding National Institutes of Health (R01AI125301)

Additional ORFs in Plant LTR-Retrotransposons.

LTR-retrotransposons share a common genomic organization in which the 5′ long terminal repeat (LTR) is followed by the gag and pol genes and terminates with the 3′ LTR. Although GAG-POL-encoded proteins are considered sufficient to accomplish the LTR-retrotransposon transposition, a number of elements carrying additional open reading frames (aORF) have been described. In some cases, the presence of an aORF can be explained by a phenomenon similar to retrovirus gene transduction, but in these cases the aORFs are present in only one or a few copies. On the contrary, many elements contain aORFs, or derivatives, in all or most of their copies. These aORFs are more frequently located between pol and 3′ LTR, and they could be in sense or antisense orientation with respect to gag-pol. Sense aORFs include those encoding for ENV-like proteins, so called because they have some structural and functional similarities with retroviral ENV proteins. Antisense aORFs between pol and 3′ LTR are also relatively frequent and, for example, are present in some characterized LTR-retrotransposon families like maize Grande, rice RIRE2, or Silene Retand, although their possible roles have been not yet determined. Here, we discuss the current knowledge about these sense and antisense aORFs in plant LTR-retrotransposons, suggesting their possible origins, evolutionary relevance, and function.

FV Vectors as Alternative Gene Vehicles for Gene Transfer in HSCs.

Hematopoietic Stem Cells (HSCs) are a unique population of cells, capable of reconstituting the blood system of an organism through orchestrated self-renewal and differentiation. They play a pivotal role in stem cell therapies, both autologous and allogeneic. In the field of gene and cell therapy, HSCs, genetically modified or otherwise, are used to alleviate or correct a genetic defect. In this concise review, we discuss the use of SFVpsc_huHSRV.13, formerly known as Prototype Foamy Viral (PFV or FV) vectors, as vehicles for gene delivery in HSCs. We present the properties of the FV vectors that make them ideal for HSC delivery vehicles, we review their record in HSC gene marking studies and their potential as therapeutic vectors for monogenic disorders in preclinical animal models. FVs are a safe and efficient tool for delivering genes in HSCs compared to other retroviral gene delivery systems. Novel technological advancements in their production and purification in closed systems, have allowed their production under cGMP compliant conditions. It may only be a matter of time before they find their way into the clinic.

Expression of the syncytin-1 and syncytin-2 genes in the trophoblastic tissue of the early pregnancy losses with normal and abnormal karyotypes.

Syncytin-1 and syncytin-2 which are endogenous retroviral genes products play a great role in syncytialization during trophoblast differentiation in normal placental tissues. In aneuploidic placentas due to the low level of pregnancy-induced hormones an alteration was occurred in the syncytialization process, while in the presence of cytogenetically abnormal karyotype the effect of syncytin gene expression levels on syncytialization process and in occured to spontaneous abortions is not clear. To reveal this, we investigated in syncytin-1 and syncytin-2 genes expression levels of chromosomally abnormal and normal trophophoblastic tissues and we also discussed the effect of the syncytin gene expression levels to the occurense of the spontaneous abortion. To each one of the trophoblastic cells; cultivation, harvesting, banding, and analysis were performed and the chromosomes were classified according to the presence of abnormality and normal XY constitution. To exclude the maternal decidual cell contamination, female karyotyped abortion materials were omitted in control group. The patient group consisted of thirty six placental tissues including trisomy 16 (n=10), triploidy (n=9), monosomyX(n=9), trisomy 21 (n=5) and trisomy 7 (n=3). The control group was consisting twenty placental tissues with XY karyotypes. The some part of the dissected frozen trophoblastic cells were used for RNA isolation and were proceeded to the determination of the expression levels of syncytin-1 and syncytin-2 genes by single-step Real Time PCR. The cDNAs were obtained by probes used in the same PCR stages. The sequence analysis of the syncytin-1 and syncytin-2 genes were performed, and read by the usage of the FinchTV 1.4.0 program. Between the expression levels of syncytin-1 and syncytin-2 genes were statistically difference in the patients and controls. There was a difference (p<0.0001) between trisomy 7 and other patient groups and controls, regarding to the expression of syncytin-1 gene. Numerous mutations in the syncytin-1 and syncytin-2 genes (on the expression sites) were detected, and the mutation rate was higher in the syncytin-1 gene compared to the syncytin-2 gene in the patient and in the control groups (p<0.001). The results of the study indicate that the expression of the syncytin-2 genes could be altered in the presence of chromosomally abnormal trophoblastic tissues, and these could lead to the loss of pregnancy due to the insufficient syncytialization. In sum, the current research has value for the further studies covering the mechanisms of trophoblast cell fusion, and syncytiotrophoblast regeneration, and thus the pathophysiology of human placental development in the presence of genomic anomaly.

DUX-miR-344-ZMYM2-Mediated Activation of MERVL LTRs Induces a Totipotent 2C-like State.

Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos, although the molecular mechanism underlying such an expanded 2CLC potency remains elusive. We examine global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We find that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with activation of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by DUX and post-transcriptionally represses ZMYM2 and its partner LSD1, and ZMYM2 recruits LSD1/HDAC corepressor complex to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the previously unappreciated DUX-miR-344-Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.

HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag.

The pol retrovirus gene encodes required enzymes for virus replication and maturation. Unlike HIV-1 Pol (expressed as a Gag–Pol fusion protein), foamy virus (described as an ancient retrovirus) expresses Pol without forming Gag–Pol polyproteins. We placed a “self-cleaving” 2A peptide between HIV-1 Gag and Pol. This construct, designated G2AP, is capable of producing virions with the same density as a wild-type (wt) HIV-1 particle. The 2A peptide allows for Pol to be packaged into virions independently from Gag following co-translationally cleaved from Gag. We found that G2AP exhibited only one-third the virus infectivity of the wt, likely due, at least in part, to defects in Pol packaging. Attenuated protease (PR) activity, or a reduction in Pol expression due to the placement of 2A-mediated Pol in a normal Gag–Pol frameshift context, resulted in significant increases in virus yields and/or titers. This suggests that reduced G2AP virus yields were largely due to increased PR activity associated with overexpressed Pol. Our data suggest that HIV-1 adopts a gag/pol ribosomal frameshifting mechanism to support virus assembly via the efficient modulation of Gag–Pol/Gag expression, as well as to promote viral enzyme packaging. Our results help clarify the molecular basis of HIV-1 gene expression and assembly.

Retroviral Gene Transduction into T Cell Progenitors for Analysis of T Cell Development in the Thymus.

The thymus is an organ where T cells develop throughout life. Using mice as a model animal, molecular mechanisms of intrathymic T cell development have been studied. Fetal thymus organ culture technique enables ex vivo reconstitution of fetal-specific T cell development, while bone marrow chimera technique allows in vivo reconstitution of T cell development in adult thymus. These techniques can be combined with retroviral gene transduction into the T cell progenitors to evaluate the function of genes of interest in developing T cells. Here, we describe the basic protocols for retrovirus gene transduction into fetal or adult T cell progenitors and reconstitution of thymic T cell development including experimental tips such as using cryopreserved fetal liver or bone marrow cells as sources of T cell progenitors.

Repurposing of retroviral genes: when foe becomes self

Throughout evolution, our genomes have been under attack by potentially harmful viruses. However, sometimes genes from the viral invaders have been captured and converted to provide a beneficial function for the host. A viral gene responsible for protecting retroviral RNA has been repurposed in both human and insect hosts to protect and transmit neurological mRNA signals. - submission by Ian A. Taylor, Jonathan P. Stoye

In Vitro Direct Reprogramming of Mouse and Human Astrocytes to Induced Neurons.

Direct neuronal reprogramming, rewiring the epigenetic and transcriptional network of a differentiated cell type to neuron, apart from being a very promising approach for the treatment of brain injury and neurodegeneration, also offers a prime opportunity to investigate the molecular underpinnings of neuronal cell fate determination, as the precise molecular mechanisms that establish neuronal fate and diversity at the transcriptional and epigenetic level are incompletely understood. Recent studies from a number of groups, including ours, have shown that astrocytes can be directly reprogrammed into functional neurons in vitro and in vivo following ectopic overexpression of combinations of transcription factors, neurogenic proteins, miRNAs, and small chemical molecules.In this chapter we describe the protocols for in vitro converting primary cortical astrocytes of mouse and human origin to induced neurons, through forced expression of two neurogenic molecules, either each one alone or in combination: the master regulatory bHLH proneural transcription factor NEUROGENIN-2 (NEUROG2) and the neurogenic protein CEND1. Forced expression of each one of the two neurogenic proteins in primary astrocytes via retroviral gene transfer results in their direct conversion to subtype-specific induced neurons, while simultaneous coexpression of both molecules drives them predominantly toward acquisition of a neural precursor cell (NPC) state. Although mouse and human astrocytes exhibit differences in their reprogramming rate and particular characteristics, they can both get efficiently in vitro transdifferentiated to NPCs and induced neurons upon NEUROG2 or/and CEND1 forced expression using the reprogramming protocols described in the chapter, presenting valuable cellular platforms for mechanistic studies and in vivo applications to restore neurodegeneration.

Abstract A062: Preclinical study of the fungal derivative cordycepin as an anti-cancer agent

Abstract Introduction: Cordycepin or 3′-deoxyadenosine is an extract from the fungus Cordyceps sinensis, a popular traditional medicine in the Far East. Although its active metabolite 3′-deoxydenosine 5′-triphosphate (3’dATP) has potent in vitro anti-cancer activity, it has shown limited efficacy in clinical trials. This is due to its poor bioavailability and rapid degradation by the ubiquitous catabolic enzyme adenosine deaminase (ADA). Here, using genetic and cellular cancer model systems, we investigated the biological impact of cordycepin in cancer cells. Experimental Procedures: Cell proliferation was measured in the presence of cordycepin and antagonists to determine its IC50. A haploid leukaemia-derived cell line, Hap1, was mutagenized using retroviral gene trapping and the mutant pool was selected for clones resistant to cordycepin which were sequenced by NGS. CRISP/Cas9 knock out combined with pharmacokinetic determination of the IC50 enabled us to validate the top hits. RNA deep sequencing-based transcriptomic analysis and gene set enrichment were used to identify the signalling pathways most affected by cordycepin and to define possible mechanisms of action. Results: We found 106 significant gene hits using the haploid genetic screen. The intracellular antiproliferative activity of cordycepin depended on both its uptake by human equilibrative nucleoside transporter 1 (hENT1) and its rate-limiting phosphorylation by the intracellular enzyme adenosine kinase (ADK). CRISPR/Cas9 knock out or selective inhibition of hENT1 or ADK led to a greater than 10-fold reduction in the activity of cordycepin. When extracellular degradation by ADA was inhibited, cordycepin showed up to 78x greater anti-proliferative potency in cancer cells. Furthermore, after 6h of treatment with cordycepin, cells showed significant differential expression of more 1000 genes. Integrating both datasets, we demonstrated by gene set enrichment analysis that, once successfully activated within the cell, 3’dATP induces cell death by a dual mechanism; by both activating the intrinsic apoptosis pathway and suppressing the TNFa-NFkB pro-survival signalling axis. Conclusions: We demonstrate here that, in the absence of ADA and in the presence of abundant hENT1 and ADK, the active metabolite of cordycepin, 3’ dATP, works as a dual driver of apoptosis in cancer cells. This suggests that a strategy of bypassing ADA and hENT1 while administering cordycepin or a chemically-modified version of cordycepin (that overcomes these resistance mechanisms) is essential to elicit any clinical anti-cancer effect. We are currently conducting a phase I trial of NUC-7738, a ProTide form of cordycepin designed to overcome these resistance mechanisms. Agents like NUC-7738 can enhance the stability and bioavailability of cordycepin, enabling it to effectively deliver its dualistic anticancer effect in patients. Citation Format: Hagen Schwenzer, Erica De Zan, Ruud van Stiphout, Cornelia H de Moor, Sebastian Nijman, Gareth Bond, Sarah P Blagden. Preclinical study of the fungal derivative cordycepin as an anti-cancer agent [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A062. doi:10.1158/1535-7163.TARG-19-A062

Intratumor Adoptive Transfer of IL-12 mRNA Transiently Engineered Antitumor CD8+ T Cells

Retroviral gene transfer of interleukin-12 (IL-12) into Tcells markedly enhances antitumor efficacy upon adoptive transfer but has clinically shown unacceptable severe side effects. To overcome the toxicity, we engineered tumor-specific CD8+ Tcells to transiently express IL-12. Engineered Tcells injected intratumorally, but not intravenously, led to complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced by co-injection with agonist anti-CD137 mAb or by transient co-expression of CD137 ligand. This treatment induced epitope spreading of the endogenous CD8+ Tcell immune response in a manner dependent on cDC1 dendritic cells. Mouse and human tumor-infiltrating Tlymphocyte cultures can be transiently IL-12 engineered to attain marked immunotherapeutic effects.