In vivo CAR T-cell generation in nonhuman primates using lentiviral vectors displaying a multidomain fusion ligand.

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo.

Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

Background: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence.

Effective, safe, and sustained correction of murine XLA using a UCOE-BTK promoter-based lentiviral vector.

X-linked agammaglobulinemia (XLA) is an immune disorder caused by mutations in Bruton's tyrosine kinase (). BTK is expressed in B and myeloid cells, and its deficiency results in a lack of mature B cells and protective antibodies. We previously reported a lentivirus (LV) BTK replacement therapy that restored B cell development and function in and double knockout mice (a phenocopy of human XLA).

Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy.

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34 cells were incubated with two successive transductions.

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs.

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

Background: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia.

Methods: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1.

Effects of a Preoperative Transdermal Fentanyl Patch on Proinflammatory Cytokine and Pain Levels During the Postoperative Period: A Randomized Controlled Trial.

Objective: The main objectives of this article were to assess the effect of preoperative transdermal fentanyl patch (TFP) on interleukin (IL)-6 and IL-8 levels and pain after laparoscopic cholecystectomy.

Materials And Methods: Patients received a TFP (25 μg/h) (patch group, n=30) or a placebo patch (control group, n=30) applied 14 hours before operation. After surgery, control group received intravenous continuous fentanyl (25 μg/h) with loading dose (25 μg).

Is the transdermal fentanyl patch an efficient way to achieve acute postoperative pain control?: A randomized controlled trial.

Backgroupd: This study investigated the plasma fentanyl concentration and efficacy of transdermal fentanyl patch (TFP) (25 μg/h) in the management of acute postoperative pain.

Methods: Patients undergoing laparoscopic cholecystectomy were randomly allocated to 2 groups. The TFP group (n = 30) received a single TFP 25 μg/ h to the anterior chest wall 14 h before operation.

Single-port laparoscopic surgery in acute appendicitis: retrospective comparative analysis for 618 patients.

Background: Transumbilical single-port laparoscopic appendectomy (SPLA) is a promising procedure that features less pain, faster recovery of postoperative bowel function and superior cosmetic results. We performed a retrospective comparative analysis of SPLA versus conventional laparoscopic surgery (CLA) to evaluate the safety and efficacy in acute appendicitis.

Methods: From December 2008 to November 2013, laparoscopic surgery was performed on 636 patients with acute appendicitis at the Department of Surgery, Chuncheon Sacred Heart Hospital.

Coupling endonucleases with DNA end-processing enzymes to drive gene disruption.

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end-processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.

Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome.

The immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to life-threatening hematopoietic cell dysfunction. We used WAS protein (WASp)-deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression.

Developmentally regulated expression of MEF2C limits the response to BCR engagement in transitional B cells.

Transitional and naïve mature peripheral B cells respond very differently to B-cell receptor (BCR) cross-linking. While transitional B cells undergo apoptosis upon BCR engagement, mature B cells survive and proliferate. This differential response correlates with the capacity of mature, but not transitional B cells to transcribe genes that promote cell survival and proliferation, including those encoding c-Myc and the Bcl-2 family members Bcl-xL and A1.

Management of endoscopic retrograde cholangiopancreatography-related perforations.

Purpose: The purpose of this study is to analyze the treatment strategies of patients with endoscopic retrograde cholangiopancreatography (ERCP)-related perforations. This is a retrospective study.

Methods: We experienced 13 perforations associated with ERCP.

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins.

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors.

Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors.

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression.

Tracking genome engineering outcome at individual DNA breakpoints.

Site-specific genome engineering technologies are increasingly important tools in the postgenomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks.

Successful targeting and disruption of an integrated reporter lentivirus using the engineered homing endonuclease Y2 I-AniI.

Current antiviral therapy does not cure HIV-infected individuals because the virus establishes lifelong latent infection within long-lived memory T cells as integrated HIV proviral DNA. Here, we report a new therapeutic approach that aims to cure cells of latent HIV infection by rendering latent virus incapable of replication and pathogenesis via targeted cellular mutagenesis of essential viral genes. This is achieved by using a homing endonuclease to introduce DNA double-stranded breaks (dsb) within the integrated proviral DNA, which is followed by triggering of the cellular DNA damage response and error-prone repair.

Acute, fatal postoperative myocardial infarction after laparoscopic cholecystectomy in a cardiac patient -A case report-.

This report presents the case of a 63-year-old man who had a myocardial infarction leading to coronary artery bypass graft 2 years earlier who subsequently underwent elective laparoscopic cholecystectomy. After an uneventful operation, the patient developed an acute postoperative myocardial infarction in the recovery room and died 19 days postoperatively. Anesthesiologists should be aware of the rare possibility of acute, fatal postoperative myocardial infarction and consider this complication when they perform the preoperative risk evaluation, anesthesia, and postoperative care for cardiac patients undergoing noncardiac surgery.

Development of B-lineage predominant lentiviral vectors for use in genetic therapies for B cell disorders.

Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin β (Igβ) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC).

B cell-specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobulinemia.

The immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy.

The block to membrane fusion differs with the site of ligand insertion in modified retroviral envelope proteins.

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env.

An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation.

Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a gamma-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2.