scholarly journals Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1

2021 ◽  
Vol 11 ◽  
Author(s):  
Anna M. D. Végh ◽  
Arie O. Verkerk ◽  
Lucía Cócera Ortega ◽  
Jianan Wang ◽  
Dirk Geerts ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Patch Clamp ◽  
Computational Modeling ◽  
Sodium Current ◽  
Pacemaker Activity ◽  
Cellular Delivery ◽  
Lentiviral Transduction ◽  
Biological Pacemaker

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a “funny current” and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

scholarly journals Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block

2014 ◽  
Vol 6 (245) ◽  
pp. 245ra94-245ra94 ◽  
Author(s):  
Yu-Feng Hu ◽  
James Frederick Dawkins ◽  
Hee Cheol Cho ◽  
Eduardo Marbán ◽  
Eugenio Cingolani
Keyword(s):  
Gene Transfer ◽  
Minimally Invasive ◽  
Heart Block ◽  
Complete Heart Block ◽  
Large Animal Model ◽  
Pacemaker Activity ◽  
Large Animal ◽  
Intramyocardial Injection ◽  
Biological Pacemaker

Somatic reprogramming by reexpression of the embryonic transcription factor T-box 18 (TBX18) converts cardiomyocytes into pacemaker cells. We hypothesized that this could be a viable therapeutic avenue for pacemaker-dependent patients afflicted with device-related complications, and therefore tested whether adenoviralTBX18gene transfer could create biological pacemaker activity in vivo in a large-animal model of complete heart block. Biological pacemaker activity, originating from the intramyocardial injection site, was evident inTBX18-transduced animals starting at day 2 and persisted for the duration of the study (14 days) with minimal backup electronic pacemaker use. Relative to controls transduced with a reporter gene,TBX18-transduced animals exhibited enhanced autonomic responses and physiologically superior chronotropic support of physical activity. Induced sinoatrial node cells could be identified by their distinctive morphology at the site of injection inTBX18-transduced animals, but not in controls. No local or systemic safety concerns arose. Thus, minimally invasiveTBX18gene transfer creates physiologically relevant pacemaker activity in complete heart block, providing evidence for therapeutic somatic reprogramming in a clinically relevant disease model.

Abstract 1744: Adenylate-Cyclase VI Transforms Ventricular Cardiomyocytes Into Biological Pacemaker Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Arjang Ruhparwar ◽  
Klaus Kallenbach ◽  
Gunnar Klein ◽  
Mahyar E Makoui ◽  
Christoph Bara ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Adenylate Cyclase ◽  
Left Ventricular ◽  
Control Group ◽  
Pacemaker Activity ◽  
Hcn Channels ◽  
Lacz Gene ◽  
Av Node ◽  
Biological Pacemaker

Background- Genetic therapy of arrhythmic disorders of the heart has been subject of extensive studies. Cyclic AMP (cAMP) is generated in response to β-adrenergic receptor stimulation and also binds to HCN channels, where it regulates spontaneous rhythmic activity in the heart. Aim of this study was to demonstrate whether in-vivo adenoviral gene transfer of Adenylate-Cyclase type VI (AC VI ) can create biological pacemaker activity in a porcine atrioventricular node (AV-node) block model. Methods and Results- Adenovirus encoding either AC VI or β-Galactosidase (lacZ) gene were injected into the lateral wall of the left ventricle of adult pigs via anterolateral thoracotomy at a dose of 10 10 virus particles each. After 12 days, the AV-node was ablated and three-dimensional electrophysiological cardiac mapping was performed using the Ensite™ electro-anatomical system. After rapid ventricular pacing and administration of Isoprenalin all animals of the AC-VI group exhibited an escape rhythm originating from the area of the left ventricular injection site at a rate of 100 ± 7 beats/minute (n = 5), while the escape rhythms in the control group (n = 4) originated from the right ventricle. Western blot analysis of the injection sites revealed a significantly higher expression of AC-VI in the respective group as compared to the control group. Conclusions- Our study demonstrates that AC-VI gene transfer may be a novel method to create a biological pacemaker

scholarly journals Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mary Jo Rademacher ◽  
Anahi Cruz ◽  
Mary Faber ◽  
Robyn A. A. Oldham ◽  
Dandan Wang ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Lines ◽  
Nk Cell ◽  
Autologous Tumor ◽  
Murine Models ◽  
Lentiviral Transduction ◽  
Ifn Γ ◽  
Human Sarcoma

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

scholarly journals Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1716
Author(s):  
Kun Tong ◽  
Ruotian Zhang ◽  
Fengzhi Ren ◽  
Tao Zhang ◽  
Junlin He ◽  
...  
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Formalin Test ◽  
Sodium Ion ◽  
Inhibitory Potency ◽  
Voltage Gated ◽  
Patch Clamp Electrophysiology ◽  
Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

scholarly journals Recapitulating Cardiac Structure and Function In Vitro from Simple to Complex Engineering

Micromachines ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 386
Author(s):  
Ana Santos ◽  
Yongjun Jang ◽  
Inwoo Son ◽  
Jongseong Kim ◽  
Yongdoo Park
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Computational Modeling ◽  
Cardiac Tissue ◽  
Cardiac Development ◽  
Heart Tissue ◽  
Cardiac Tissue Engineering ◽  
Cardiac Cells

Cardiac tissue engineering aims to generate in vivo-like functional tissue for the study of cardiac development, homeostasis, and regeneration. Since the heart is composed of various types of cells and extracellular matrix with a specific microenvironment, the fabrication of cardiac tissue in vitro requires integrating technologies of cardiac cells, biomaterials, fabrication, and computational modeling to model the complexity of heart tissue. Here, we review the recent progress of engineering techniques from simple to complex for fabricating matured cardiac tissue in vitro. Advancements in cardiomyocytes, extracellular matrix, geometry, and computational modeling will be discussed based on a technology perspective and their use for preparation of functional cardiac tissue. Since the heart is a very complex system at multiscale levels, an understanding of each technique and their interactions would be highly beneficial to the development of a fully functional heart in cardiac tissue engineering.

scholarly journals An approach for the analysis of relapse and marrow reconstitution after autologous marrow transplantation using retrovirus-mediated gene transfer

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2694-2700 ◽  
Author(s):  
DR Rill ◽  
RC Moen ◽  
M Buschle ◽  
C Bartholomew ◽  
NK Foreman ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Marrow Transplantation ◽  
Residual Disease ◽  
Marker Gene ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Disease Recurrence ◽  
Malignant Cells

Abstract Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested “remission” marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.

scholarly journals Gene transfer of multidrug resistance into a factor-dependent human hematopoietic progenitor cell line: in vivo model for genetically transferred chemoprotection

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2723-2731 ◽  
Author(s):  
P Schwarzenberger ◽  
S Spence ◽  
N Lohrey ◽  
T Kmiecik ◽  
DL Longo ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Gene Transfer ◽  
Hematopoietic Progenitor Cells ◽  
Mdr1 Gene ◽  
In Vivo Model ◽  
Hematopoietic Progenitor ◽  
Human Dna

To develop a rapid preclinical in vivo model to study gene transfer into human hematopoietic progenitor cells, MO-7e cells (CD-34+, c-kit+) were infected with multidrug resistance (MDR1)-containing retroviruses and then transplanted into nonobese diabetic severe combined immunodeficient mice (NOD SCID). MO-7e cells infected with a retrovirus encoding the human MDR1 cDNA showed integration, transcription, and expression of the transfered MDR1 gene. This resulted in a 20-fold increase in the resistance of MO-7e cells to paclitaxel in vitro. The expression of the MDR1 gene product was stable over a 6-month period in vitro without selection in colchicine. MO-7e and MDR1-infected MO-7e cells were transplanted into NOD SCID mice to determine whether MDR1 could confer drug resistance in vivo. A sensitive polymerase chain reaction method specific for human sequences was developed to quantitate the level of human cell engraftment in NOD SCID bone marrow (BM) cells. The percentage of human DNA in BM cells from MO-7e- transplanted mice was 10.9% and decreased to 0.7% in mice treated with paclitaxel. The percentage of human DNA in infected-MO-7e transplanted mice was 7.6% and that level was unchanged in mice treated with paclitaxel. These results show that expression of the MDR1 gene in human hematopoietic progenitor cells can confer functional drug resistance in an in vivo model.

Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene™6: in vitro and in vivo studies

2000 ◽  
Vol 57 (8) ◽  
pp. 1326-1333 ◽  
Author(s):  
I. Hellgren* ◽  
V. Drvota ◽  
R. Pieper ◽  
S. Enoksson ◽  
P. Blomberg ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Viral Vectors ◽  
In Vivo Studies ◽  
Highly Efficient
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