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Objective: This paper aims to review the risks associated with using lentiviral and retroviral vectors in research and clinical settings and to propose an update to an effective treatment plan. Methods: Risks of exposure were evaluated based on vector design, safety features, viral tropism, transgene, and means and modes of transmission. These risks wereweighed against the potential risks and benefits of current HIV medications. Results:We recommend the following postexposure prophylactic treatment for significant lentiviral vector exposures: 1) dolutegravir 50 mg taken once a day for 7 days and 2) tenofovir disoproxil fumarate 300 mg taken once a day for 7 days (28 days of both medications for replication-competent vectors). Conclusions: Because of the highly efficient delivery of transgenes by modern lentiviral and retroviral vectors, postexposure prophylaxis is indicated to prevent vector integration and oncogenic risks. [ABSTRACT FROM AUTHOR]

To construct SW620 cell line expressing human CD19, enhanced Green fluorescent protein (EGFP) and luciferase (LUC). Construct the MFG-CD19 plasmid, package it with a retroviral vector, and co transfect it with theMFG-EGFP-P2A-LUC retroviral vector to SW620 cells. Select a stable and strongly expressed monoclonal cell lineSW620-CD19-EGFP-LUC and expand the culture. The expression of CD19 on the cell was detected by flow cytometry, and the expression of luciferase on the cell line surface and cell line function were identified by luciferase assay. The percentage of CD19-positive cells and EGFP-positive cells was 99.9% and 99.2%, respectively, and the green fluorescence could be observed in the Fluorescence microscope The results of qPCR showed that the expression level of CD19 in the cell lines was significantly up-regulated compared with the original cell lines. The SW620 cell line that can stably express human CD19 and fluorescent protein was successfully constructed through retroviral vector packaging technology. [ABSTRACT FROM AUTHOR]

Simple Summary: Cells from the malignant brain tumor, glioblastoma multiforme (GBM) are highly heterogeneous. After tumor removal, some tumor cells remain at the tumor-brain boundary since in the brain, surgery cannot be performed with the normally required safety margin. Thus, it is of upmost importance to develop tools able to destroy remaining tumor cells. One strategy is to develop lentiviral vectors (LVs) with high specificity for GBM cells to transfer therapeutic genes into these cells. The Zika virus (ZIKV) provides an envelope with the protein E, which has a high specificity for GBM cells, making it a prime candidate for the development of LVs; so-called ZIKV protein E coated lentiviral particles. The study demonstrates that such LVs have an efficiency and high specificity for GBM tumor cells, leaving healthy cells mostly unharmed. These LVs open up new perspectives and therapeutic options for combating tumor cells that cannot be removed through surgery. The development of new tools against glioblastoma multiforme (GBM), the most aggressive and common cancer originating in the brain, remains of utmost importance. Lentiviral vectors (LVs) are among the tools of future concepts, and pseudotyping offers the possibility of tailoring LVs to efficiently transduce and inactivate GBM tumor cells. Zika virus (ZIKV) has a specificity for GBM cells, leaving healthy brain cells unharmed, which makes it a prime candidate for the development of LVs with a ZIKV coat. Here, primary GBM cell cultures were transduced with different LVs encased with ZIKV envelope variants. LVs were generated by using the pNLgfpAM plasmid, which produces the lentiviral, HIV-1-based, core particle with GFP (green fluorescent protein) as a reporter (HIVgfp). Using five different GBM primary cell cultures and three laboratory-adapted GBM cell lines, we showed that ZIKV/HIVgfp achieved a 4–6 times higher transduction efficiency compared to the commonly used VSV/HIVgfp. Transduced GBM cell cultures were monitored over a period of 9 days to identify GFP+ cells to study the oncolytic effect due to ZIKV/HIVgfp entry. Tests of GBM tumor specificity by transduction of GBM tumor and normal brain cells showed a high specificity for GBM cells. [ABSTRACT FROM AUTHOR]

Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990-2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription ("SIN" vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs.

A fundamental idea for targeting glioblastoma cells is to exploit the neurotropic properties of Zika virus (ZIKV) through its two outer envelope proteins, prM and E. This study aimed to develop envelope glycoproteins for pseudotyping retroviral vectors that can be used for efficient tumor cell infection. Firstly, the retroviral vector pNLlucAM was packaged using wild-type ZIKV E to generate an E-HIVluc pseudotype. E-HIVluc infection rates for tumor cells were higher than those of normal prME pseudotyped particles and the traditionally used vesicular stomatitis virus G (VSV-G) pseudotypes, indicating that protein E alone was sufficient for the formation of infectious pseudotyped particles. Secondly, two envelope chimeras, E41.1 and E41.2, with the E wild-type transmembrane domain replaced by the gp41 transmembrane and cytoplasmic domains, were constructed; pNLlucAM or pNLgfpAM packaged with E41.1 or E41.2 constructs showed infectivity for tumor cells, with the highest rates observed for E41.2. This envelope construct can be used not only as a tool to further develop oncolytic pseudotyped viruses for therapy, but also as a new research tool to study changes in tumor cells after the transfer of genes that might have therapeutic potential. [ABSTRACT FROM AUTHOR]

Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.

To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale.

Treatment of tumors with Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, followed by 5-fluorocytosine (5-FC) kills tumors by local production of 5-fluorouracil (5-FU). In brain tumor models, this treatment induces systemic anti-tumor immune responses and long-term immune-mediated survival. Phase 1 Toca 511 and Toca FC (extended-release 5-FC) clinical trials in patients with recurrent high-grade glioma show durable complete responses and promising survival data compared to historic controls. The work described herein served to expand on our earlier findings in two models of metastatic colorectal carcinoma (mCRC). Intravenous (i.v.) delivery of Toca 511 resulted in substantial tumor-selective uptake of vector into metastatic lesions. Subsequent treatment with 5-FC resulted in tumor shrinkage, improved survival, and immune memory against future rechallenge with the same CT26 CRC cell line. Similar results were seen in a brain metastasis model of mCRC. Of note, 5-FC treatment resulted in a significant decrease in myeloid-derived suppressor cells (MDSCs) in mCRC tumors in both the liver and brain. These results support the development of Toca 511 and Toca FC as a novel immunotherapeutic approach for patients with mCRC. A phase 1 study of i.v. Toca 511 and Toca FC in solid tumors, including mCRC, is currently underway (NCT02576665).

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes, or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency, and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild-type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

Two patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency (ADA-SCID) received stem cell-based gene therapy (SCGT) using GCsapM-ADA retroviral vectors without preconditioning in 2003 and 2004. The first patient (Pt1) was treated at 4.7 years old, and the second patient (Pt2), who had previously received T cell gene therapy (TCGT), was treated at 13 years old. More than 10 years after SCGT, T cells showed a higher vector copy number (VCN) than other lineages. Moreover, the VCN increased with differentiation toward memory T and B cells. The distribution of vector-marked cells reflected variable levels of ADA requirements in hematopoietic subpopulations. Although neither patient developed leukemia, clonal expansion of SCGT-derived clones was observed in both patients. The use of retroviral vectors yielded clonal dominance of vector-marked clones, irrespective of the lack of leukemic changes. Vector integration sites common to all hematopoietic lineages suggested the engraftment of gene-marked progenitors in Pt1, who showed severe osteoblast (OB) insufficiency compared to Pt2, which might cause a reduction in the stem/progenitor cells in the bone marrow (BM). The impaired BM microenvironment due to metabolic abnormalities may create space for the engraftment of vector-marked cells in ADA-SCID, despite the lack of preconditioning.

Objective To construct a retroviral vector-mediated SynNotch gating system. Methods The retroviral vector was designed and modified，insulator sequences were inserted at both ends，and the SynNotch gating system was constructed based on the Gal4/UAS gene expression regulation system. First，a retroviral vector was used to construct a gated plasmid containing only Gal4-VP64 fusion protein transcriptional regulatory elements and an effector plasmid contain⁃ ing the UAS activation sequence. The effector plasmids used green fluorescent protein（EGFP）and luciferase as reporter genes，and were divided into 6 types，three of which were designed for the forward sequence，and the UAS sequence and the reporter gene were inserted into the retroviral vector（5'-3'）in the forward direction，including the forward non-insula⁃ tor，forward single insulator，and forward double insulator，respectively；the other three were designed for reverse se⁃ quence，and the UAS sequence and the reporter gene were reversely inserted into the retroviral vector（3'-5'），including reverse non-insulator，reverse single insulator，and reverse double insulator，respectively. After the plasmid was construct⁃ ed，it was verified by sequencing，and the successfully sequenced plasmid was further prepared by virus packaging to pre⁃ pare viral vectors，including gated vectors and effector vectors. PCR method was used to detect retrovirus titers. The viral vector was transduced into 293T cells，the 293T cells transduced by the effector vector alone were used as the uninduced group，and the 293T cells co-transduced by the gated vector and the effector vector were used as the induced group. Flow cytometry and luciferase chemiluminescence experiment were used to detect the expression efficiency of reporter genes in the two groups. Results The titers of retroviral vectors were 107 -108 copies/mL，and were not lower than those of the pos⁃ itive control. The results of flow cytometry showed that the expression efficiencies of EGFP were 95. 5%，69. 7%，and 76. 2% in the three forwardly designed effector vectors without induction，and the expression efficiencies of EGFP after in⁃ duction were 99. 9% ，95. 3%，and 97. 1%，respectively. The three reversely designed effector vectors had EGFP expres⁃ sion efficiencies of 0. 085%，0. 61%，and 7. 4% without induction，and EGFP expression efficiencies of 59. 9%，77. 9%， and 89. 5% after induction. The detection results of the luciferase chemiluminescence experiment showed that the fluores⁃ cence intensity of luciferase expression of the three effect vectors in the forward design was above 104 without induction， and the fluorescence intensity after induction was above 105 . The fluorescence intensity of luciferase expressed by the effec⁃ tor vector was below 3×103 without induction，and the fluorescence intensity after induction was above 104 . The fluores⁃ cence intensity after induction was significantly different from that before induction（P<0. 01）. Among them，the fluores⁃ cence intensity after induction by the reverse double insulator design reached more than 105 . Conclusion A retroviral vector-mediated SynNotch gating system was successfully constructed in this study. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cells targeting CD19 antigen have produced remarkable clinical outcomes for cancer patients. However, identifying measures to enhance effector function remains one of the most challenging issues in CD19-targeted immunotherapy. Here, we report a novel approach in which a microRNA (miRNA) or short-hairpin RNA (shRNA) cassette was integrated into CAR-expressing retroviral vectors. Using this system, we generated anti-CD19 CAR-T cells co-expressing miR155 or LSD1 shRNA and found that anti-CD19 CAR-T cells with miR155 upregulation or LSD1 downregulation exhibited increased anti-tumor functions in vitro and in vivo. Transcriptional profiling analysis by RNA sequencing revealed the targets of miR155 and LSD1 in anti-CD19 CAR-T cells. Our experiments indicated that introduction of miRNA or shRNA expression into anti-CD19 CAR T-cells might be an effective strategy to improve the anti-tumor effects of CAR-T cell therapy.

Self‐assembling peptide nanofibrils (PNF) have gained increasing attention as versatile molecules in material science and biomedicine. One important application of PNF is to enhance retroviral gene transfer, a technology that has been central for gene therapy approaches. The best‐investigated and commercially available PNF is derived from a 12‐mer peptide termed EF‐C. The mechanism of transduction enhancement depends on the polycationic surface of EF‐C PNF, which bind to the negatively charged membranes of viruses and cells, thereby overcoming electrostatic repulsions and increasing virion attachment and fusion. To better understand how EF‐C PNF interact with the cell surface, scanning electron and time‐lapse confocal microscopy were performed. The fibrils are found to be actively engaged by cellular protrusions such as filopodia. Consequently, chemical suppression of protrusion formation abrogates fibril binding and virion delivery to the cell surface of immortalized and primary T cells. Vice versa, induction of plasma membrane blebs result in increased fibril binding. Thus, the mechanism of PNF‐mediated viral transduction enhancement involves an active engagement of virus‐loaded fibrils by cellular protrusions, which may explain its superior performance over soluble transduction enhancers. [ABSTRACT FROM AUTHOR]

• Gene therapy represents a viable treatment option for various diseases. • Clinical trials have been conducted on gene therapy addressing genodermatoses. • This review summarizes the current use of gene therapy in the dermatological field. • Superficial gene-therapy sites can be evaluated for presence of neoplasms. • Therefore, dermatologists have an important role in gene therapy application. With recent advances in genetic engineering technology, gene therapy is now being considered as a treatment not only for congenital diseases but also acquired diseases, such as cancer. Gene therapeutic agents for hereditary immune disorders, haemophilia, retinal diseases, neurodegenerative diseases, and lymphoma have been approved in the United States and Europe. In the field of dermatology, clinical trials of gene therapy have been conducted, because the skin is an easily accessible organ that represents an attractive tissue for gene therapy. In recent years, gene therapy has been attempted for a variety of skin diseases, such as genodermatoses (including epidermolysis bullosa and Netherton syndrome), cutaneous lymphoma, and malignant melanoma. As a result, it is difficult to grasp the current status of gene therapy in dermatology. This review focuses on each of the gene-transfer techniques currently in use and describes the current status of gene therapy for skin diseases using each technology. [ABSTRACT FROM AUTHOR]

The human APOBEC3A (A3A) polynucleotide cytidine deaminase has been shown to have antiviral activity against HTLV-1 but not HIV-1, when expressed in the virus producer cell. In viral target cells, high levels of endogenous A3A activity have been associated with the restriction of HIV-1 during infection. Here we demonstrate that A3A derived from both target cells and producer cells can block the infection of Moloney-MLV (MLV) and related AKV-derived strains of MLV in a deaminase-dependent mode. Furthermore, glycosylated Gag (glycoGag) of MLV inhibits the encapsidation of human A3A, but target cell A3A was not affected by glycoGag and exerted deamination of viral DNA. Importantly, our results clearly indicate that poor glycoGag expression in MLV gag-pol packaging constructs as compared to abundant levels in full-length amphotropic MLV makes these viral vectors sensitive to A3A-mediated restriction. This raises the possibility of acquiring A3A-induced mutations in retroviral gene therapy applications. [Display omitted] • The interferon-inducible A3A in human macrophages can restrict incoming MLV. • A3A incorporates into MLV cores and inhibits MLV propagation. • The enzymatic activity of A3A is crucial for MLV inhibition. • Glycosylated Gag expression blocks A3A packaging into MLV. • Glycosylated Gag does not counteract target cell A3A. [ABSTRACT FROM AUTHOR]

Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990–2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription (“SIN” vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs.

Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990–2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription ("SIN" vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs. [ABSTRACT FROM AUTHOR]

Objective · To construct retroviral small guide RNA (sgRNA) expression vector for studying gene function in mouse T cells. Methods · The short hairpin RNA (shRNA)-expressing retroviral vector MSCV-LTR-miR30-PIG (LMP) was digested with Xho1 and Sal1 to obtain the backbone of the retroviral vector. The lentiviral sgRNA vector was used as template for PCR amplification to obtain the U6-sgRNA-PGK-Puro-BFP fragment. The PCR fragment was then assembled into the retroviral vector backbone by homologous recombination. To verify the effectiveness of the system, sgRNA sequences targeting Il17a, Rorc and Irf4 were designed and cloned. CD4 T cells isolated from Cas9 transgenic mice were infected with individual sgRNA retrovirus and differentiated into Th17 cells. After cytokine intracellular staining, the percentage of Il17a positive population was detected by flow cytometry. Unpaired t test was used to compare the data of independent samples between the two groups. Results · The sgRNA retroviral vector was successfully constructed. The retroviral vector was used to successfully deliver sgRNA to CD4 T cells, which mutated Il17a gene. The percentage of Il17a positive population was significantly reduced in the Rorc-sgRNA and Irf4-sgRNA positive populations (P<0.05). Conclusion · The sgRNA retroviral vector is successfully used to deliver sgRNA to target cells. In addition, the results of Rorc and Irf4 gene knockout experiments prove that the system can be used for studying gene function in mouse T cells. [ABSTRACT FROM AUTHOR]

Viral vector–mediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. We compared multiple commercially available transfection reagents to determine which was optimal for retroviral infections of primary myoblasts. Centrifugation force was also tested, and a spin infection protocol with centrifugation at 2800 × g for 90 min had the highest infection efficiency for primary myoblasts. We confirmed that infected muscle stem cells maintain cell proliferation and the capacity for in vitro and in vivo myogenic differentiation. Our new, efficient retroviral infection protocol for muscle stem cells can be applied to molecular biology experiments as well as translational studies.

Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty -derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 106 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector. [ABSTRACT FROM AUTHOR]

Sequencing data from large cohorts of T-cell acute lymphoblastic leukemia patients identified a significant association between the presence of JAK3 mutations and ectopic HOXA9 expression. Mouse models using a constitutive or novel inducible retroviral expression vector to express the JAK3(M511I) mutant and HOXA9 led to the development of an aggressive leukemia in vivo, with shorter latency than JAK3(M511I) or HOXA9 alone. This was primarily due to the co-binding of STAT5 and HOXA9 to the same genomic loci leading to increased oncogenic JAK-STAT signaling.

Abstract Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE®'s) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site. In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE®'s derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth. [ABSTRACT FROM AUTHOR]

Animal bioreactors are genetically modified animal systems that have the potential to reduce production cost, and improve production efficiency, of pharmaceutically relevant recombinant proteins. Several species including goats, cattle, rabbits, and avians have been genetically modified to secrete target proteins into milk, egg whites, blood, or other bodily fluids. There are several advantages associated with the use of avians as bioreactor systems. Avians have a short generation time, leading to the quick establishment of a transgenic line and high egg production. Transgenic avian systems allow for appropriate post-translational modification, as opposed to prokaryotic cell culture bioreactors, and have higher productivity than mammalian cell culture systems. Furthermore, recombinant proteins can be incorporated into egg whites and easily collected from the sterile environment of the egg. Magnum-specific expression of target genes has been achieved by use of the ovalbumin promoter, leading to a localization of the target protein into the avian egg. In this review, we discuss the current advancements, future potential, and limitations of avian bioreactor systems. [ABSTRACT FROM AUTHOR]

Abstract A replication-deficient lentiviral vector encoding the tumor antigen gene NY-ESO-1 was characterized in terms of vector morphology, particle size range, concentration, and zeta potential using a variety of physical methods. Environmentally stressed vector samples were then evaluated in terms of viral vector particle size and concentration by nanoparticle tracking analysis (NTA). These NTA stability results correlated reasonably well with a quantitative polymerase chain reaction assay for quantitation of viral genome copy number (r2 = 0.80). Approximately 40 pharmaceutical excipients were examined for their ability to stabilize the vector against exposure to an adsorptive container surface (glass) as well as freeze-thaw cycling using NTA as the screening method. Stabilizing additives that inhibited viral vector particle loss under these conditions included proline, lactose, and mannitol. Several candidate frozen liquid formulations that contained a combination of these lead excipients and various buffering agents were further evaluated for their ability to stabilize the viral vector. The additional benefit of lowering the Tris buffer concentration was observed. This study highlights the use of physical particle assays such as NTA for initial screening of stabilizing excipients to minimize vector loss due to container adsorption and freeze-thaw cycling to facilitate early formulation development of viral vector candidates in frozen liquid formulations. [ABSTRACT FROM AUTHOR]

Adoptive T-cell therapy is being considered as a promising method for cancer treatment. In this approach, patient’s T cells are isolated, modified, expanded, and administered back to the patient. Modifications may include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) to the isolated cells by using retroviral vectors. PG13 cells, derivatives of NIH3T3 mouse fibroblasts, are being used to stably produce retroviral vectors that transduce the T cells. PG13 cells are anchorage-dependent cells that grow in roller bottles or cell factories and lately also in fixed bed bioreactors to produce the needed viral vector. To scale up viral vector production, PG13 cells were propagated on microcarriers in a stirred tank bioreactor utilizing an alternating tangential flow perfusion system. Microcarriers are 10 μm–0.5 mm beads that support the attachment of cells and are suspended in the bioreactor that provides controlled growth conditions. As a result, growth parameters, such as dissolved oxygen concentration, pH, and nutrients are monitored and continuously controlled. There were no detrimental effects on the specific viral vector titer or on the efficacy of the vector in transducing the T cells of several patients. Viral vector titer increased throughout the 11 days perfusion period, a total of 4.8 × 10 11 transducing units (TU) were obtained with an average titer of 4.4 × 10 7  TU/mL and average specific productivity of 10.3 (TU) per cell, suggesting that this method can be an efficient way to produce large quantities of active vector suitable for clinical use. [ABSTRACT FROM AUTHOR]

To identify the importance of IRAK2 kinase activity in TLR-mediated signaling pathways, we constructed a retroviral vector harboring either a mouse IRAK2 gene (IRAK2-WT) or with its mutant with loss of function of its ATP-binding site (IRAK2-KD). Further, we comparatively analyzed for the gain of function and modulations in TLR-mediated signaling pathways in IRAK2 knockout (IRAK2-KO) macrophages upon introduction of the IRAK2-WT retroviral constructs. The pBS/IRAK2-KD with the ATP-binding site mutation in IRAK2 was obtained by using site-specific mutagenesis. The recombinants were identified with appropriate double digestion and sequence analysis. The recombinant vector constructs were transfected by lipofection into phoenix packaging cells. The viral vectors (107 cfu/mL) with the construct were allowed to infect IRAK2-KO macrophages. The results showed that IRAK2-WT gene overexpressed in the IRAK2-KO macrophages exhibited a modified IRAK2 expression upon LPS induction. However, the modification was absent with IRAK2-KD construct on LPS stimulation; instead, the IRAK2 protein stability was reduced considerably. The results further show that the LPS-induced effect on the stability of IRAK2 is dependent of IRAK4 stimulation. [ABSTRACT FROM AUTHOR]

Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins.

Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved.

Hypervascular hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality. Angiogenesis is an important contributor to HCC progression and metastasis; therefore, inhibiting angiogenesis may be an effective method of treating HCC. Tumstatin is a novel type of efficient endogenous vascular endothelial cell growth inhibiting factor. The anti-angiogenic activity of tumstatin is localized to the 54-132 amino acid region (Tum-5). In a previous study performed by our group, the gene fragment encoding Tum-5 was cloned and inserted into a pLXSN retroviral vector. In the present study, the anti-angiogenic effects of Tum-5 and the antitumor effects exerted by the pLXSN-Tum-5 vector in vivo were investigated. The results demonstrated that pLXSN-Tum-5 significantly inhibited the growth of human umbilical vein endothelial cells compared with pLXSN, but had no obvious effect on HepG2 cell growth. Moreover, the antitumor and anti-angiogenic activity of Tum-5 was examined in vivo using a xenograft of H22 HCC cells. The results indicated that pLXSN-Tum-5 significantly inhibited tumor growth following 5 injections over 10 days. The size and weight of tumors in the pLXSN-Tum-5 group were lower than those in the saline and pLXSN groups. Furthermore, immunohistochemical analysis with CD31 antibodies indicated that the average microvessel density in the pLXSN-Tum-5 group were significantly lower than that in the saline and pLXSN groups. These results suggested that Tum-5 exerts its antitumor activity by suppressing vascular endothelial cells. The gene fragment of Tum-5 may be developed as an effective inhibitor of angiogenesis and used to treat patients with HCC. [ABSTRACT FROM AUTHOR]

Stem cell gene therapy using the MFGS-gp91 phox retroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient's refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6 months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOM locus was identified in blast cells. The vector integration into MECOM was detectable in most myeloid cells at 12 months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1 tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications., Competing Interests: JM is inventor on patents that claim to use peptide nanofibrils as transduction enhancer. JM, DS, LR-W, KK and TW filed additional patents to use peptides as transduction enhancer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rauch-Wirth, Renner, Kaygisiz, Weil, Zimmermann, Rodriguez-Alfonso, Schütz, Wiese, Ständker, Weil, Schmiedel and Münch.)

Retroviruses cause cancers in a variety of animals and humans. Research on retroviruses has provided important insights into mechanisms of oncogenesis in humans, including the discovery of viral oncogenes and cellular proto-oncogenes. The subject of this review is the mechanisms by which retroviruses that do not carry oncogenes (non-acute retroviruses) cause cancers. The common theme is that these tumors result from insertional activation of cellular proto-oncogenes by integration of viral DNA. Early research on insertional activation of proto-oncogenes in virus-induced tumors is reviewed. Research on non-acute retroviruses has led to the discovery of new proto-oncogenes through searches for common insertion sites (CISs) in virus-induced tumors. Cooperation between different proto-oncogenes in development of tumors has been elucidated through the study of retrovirus-induced tumors, and retroviral infection of genetically susceptible mice (retroviral tagging) has been used to identify cellular proto-oncogenes active in specific oncogenic pathways. The pace of proto-oncogene discovery has been accelerated by technical advances including PCR cloning of viral integration sites, the availability of the mouse genome sequence, and high throughput DNA sequencing. Insertional activation has proven to be a significant risk in gene therapy trials to correct genetic defects with retroviral vectors. Studies on non-acute retroviral oncogenesis provide insight into the potential risks, and the mechanisms of oncogenesis.

Epidermal growth factor (EGF), which promotes epidermal regeneration and wound closure, is important for the proliferation and differentiation of epidermal and epithelial tissues in animals. Exogenous EGF is a promising therapeutic agent for wound healing, but its general use is restricted by the limited availability of this protein. In this work, we show that the transfection of mouse BALB/MK keratinocytes, which are totally dependent on EGF for growth and migration, with mature cDNA for human EGF via a retroviral vector abolished the cells requirement for exogenous EGF. The transformed cells had normal morphology and a growth rate that varied according to the source of the retroviral vector used. Keratinocyte transfection with EGF cDNA provides a time- and cost-efficient means of culturing keratinocytes and yields cells that may be useful for skin grafting.

To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat.SE in male Sprague-Dawley rats lasting for more than 2 hours was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born five days after SE were studied at least 10 weeks after CAG-GFP retroviral vector-mediated labeling.Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or after being activated by transient seizure activity than vicinal GFP-unlabeled granule cells.Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells.

The pantropic retroviral expression system has been widely used to stably introduce foreign genes into the genome of dividing cells from non-mammalian systems. However, we found that the current commercial retrovirus expression system did not work in inactively dividing primary shrimp cells. To overcome this, we successfully developed a triple-pseudotyped retroviral expression system that can be used for effective gene transfer and expression of reporter genes in both mammalian and penaeid shrimp cells by improving the commercial pantropic retrovirus system (VPK-305, Cell Biolabs) in two ways. First, the promoter regions of translationally controlled tumor protein (TCTP) genes were cloned from the shrimps Metapenaeus ensis and Marsupenaeus japonicus , named P me-tctp and P mj-tctp , respectively. We inserted them into the retroviral reporter vectors of pMCs-P LTR -GFP or eGFP, immediately after the retroviral promoter P LTR (5′LTR, long terminal repeat sequence). We then analyzed their expression efficiencies in mammalian and shrimp cells by the lipofection method. It was found that P LTR was a strong promoter in mammalian cells, but failed to effectively drive the reporter genes to express in the primary lymphoid cells (LCOO) derived from Oka organs of shrimp ( M. ensis ). However, inclusion of the shrimp promoters (P me-tctp and P mj-tctp ) significantly improved the expression efficiency of the retroviral vectors in shrimp cells. Next, the revised retroviral vectors of pMCs-P LTR -P me-tctp (or P mj-tctp )-GFP or eGFP were pseudotyped with envelope protein VSV-G alone, or triply with VSV-G, VP19 and VP28 (two envelope proteins of shrimp white spot syndrome virus). And we then examined the corresponding infection and expression efficiencies in shrimp primary LCOO cells and embryo cells. We found that only the triple-pseudotyped retrovirus containing the shrimp-derived promoter could successfully mediate the delivery and fluorescent expression of GFP and eGFP genes, but VSV-G-pseudotyped retrovirus had poor tropism to shrimp LCOO cells. Confocal microscopic observation further confirmed the hypothesis that the improved tropism of the triple-pseudotyped retrovirus to shrimp cells might be attributed to the inclusion of VP19 and VP28 into the envelope of the packaged retrovirus. In spite of this, the expression efficiency and infection efficiency (20%–30%) of reporter genes in shrimp cells was still lower than those in mammalian cells. In infected shrimp primary embryo cells, strong green fluorescent signal was detected only in neuron-like cells, not in fibroblast-like cells. This triple-pseudotyped retroviral expression system ultimately may prove useful for immortalization of cultured shrimp cells and production of transgenic shrimps. Statement of relevance • We developed a novel triple-pseudotyped retroviral expression system that can be used in effective gene transfer and expression of foreign genes in shrimp cells. • This system may prove useful for the immortalization of cultured shrimp cells which are needed in the study and control of shrimp viral diseases. • This system may prove useful for the production of transgenic shrimps with new traits. [ABSTRACT FROM AUTHOR]