Abstract 2882: Directed Evolution of Cardiac Specific Adeno-Associated Vector Variants

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Roger J Hajjar ◽  
Jiqiu Chen ◽  
Yoshiaki Kawase ◽  
Scott McPhee ◽  
Jade Samulski ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Neutralizing Antibodies ◽  
Cardiac Tissue ◽  
Marker Gene ◽  
Green Fluorescence ◽  
Viral Genomes ◽  
Large Animals ◽  
Time Point

Adeno-associated vectors (AAV) have gained popularity in cardiovascular gene transfer because of their distinct characteristics: long-term expression, minimal immune response and strong tropism to cardiac tissue for specific serotypes (such as AAV9). However AAVs have certain limitations: Neutralizing antibodies in 40% of humans for each AAV serotype and non-specificity of the AAV serotypes to various organs. Chimeric viruses composed of capsid proteins of different serotypes, show distinct transduction profiles. To identify hybrid viruses that can selectively transduce cardiomyocytes in vivo, we generated a library of diverse AAV variants, obtained by DNA shuffling, with an enrichment of cardiotropic AAV variants, called biological nanoparticles (BNPs). We selected five BNPs from these experiments: BNP 108, 109, 111, 689, and 693. We then injected these different BNPs using GFP as a marker gene under the control of CMV along with AAV9 into rats (3 in each group and each time point) through their tail vein at a concentration of 1011 vg (viral genomes). We sacrificed the animals at one and four weeks and examined expression in the heart, liver, lung, kidneys, and aorta. We found that at 1 week, BNP 108 and 109 had the best expression in the heart (61±12% of the myocytes with green fluorescence) while BNP 111 and AAV9 had strong cardiac expression at 4 weeks (93±11% and 79±12% of the myocytes with green fluorescence). None of the BNPs showed expression in the liver, lung, kidneys, and aorta, while AAV9 demonstrated widespread expression in all these tissues. Transcoronary injection of BNP111 at a concentration of 1012 vg demonstrated the efficiency of BNP in transducing myocardium of large animals. We also found that all the BNPs are resistant to preexisting neutralizing antibodies against parent serotypes. These results show that directed evolution of AAV variants can yield BNPs that are highly cardiotropic and highly efficient of transducing cardiac tissue without infecting other organs and having resistance to preexisting neutralizing antibodies against parent serotypes. These BNPs may become very useful for clinical applications.

scholarly journals Assessment of Electrospun Pellethane-Based Scaffolds for Vascular Tissue Engineering

Materials ◽  
2021 ◽  
Vol 14 (13) ◽  
pp. 3678
Author(s):  
Vera Chernonosova ◽  
Alexandr Gostev ◽  
Ivan Murashov ◽  
Boris Chelobanov ◽  
Andrey Karpenko ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
Ex Vivo ◽  
Vascular Grafts ◽  
Wistar Rat ◽  
Graft Occlusion ◽  
Suitable Candidate ◽  
Large Animals ◽  
Cell Adhesion And Proliferation

We examined the physicochemical properties and the biocompatibility and hemocompatibility of electrospun 3D matrices produced using polyurethane Pellethane 2363-80A (Pel-80A) blends Pel-80A with gelatin or/and bivalirudin. Two layers of vascular grafts of 1.8 mm in diameter were manufactured and studied for hemocompatibility ex vivo and functioning in the infrarenal position of Wistar rat abdominal aorta in vivo (n = 18). Expanded polytetrafluoroethylene (ePTFE) vascular grafts of similar diameter were implanted as a control (n = 18). Scaffolds produced from Pel-80A with Gel showed high stiffness with a long proportional limit and limited influence of wetting on mechanical characteristics. The electrospun matrices with gelatin have moderate capacity to support cell adhesion and proliferation (~30–47%), whereas vascular grafts with bivalirudin in the inner layer have good hemocompatibility ex vivo. The introduction of bivalirudin into grafts inhibited platelet adhesion and does not lead to a change hemolysis and D-dimers concentration. Study in vivo indicates the advantages of Pel-80A grafts over ePTFE in terms of graft occlusion, calcification level, and blood velocity after 6 months of implantation. The thickness of neointima in Pel-80A–based grafts stabilizes after three months (41.84 ± 20.21 µm) and does not increase until six months, demonstrating potential for long-term functioning without stenosis and as a suitable candidate for subsequent preclinical studies in large animals.

Abstract 89: MitoTimer Mouse: a Novel Tool for the Study of Mitochondrial Turnover in vivo

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Aleksandr B Stotland ◽  
Jennifer Ramil ◽  
Roberta A Gottlieb
Keyword(s):  
Cardiac Tissue ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Green Fluorescence ◽  
Physiological Conditions ◽  
High Membrane Potential ◽  
Mitochondrial Turnover ◽  
Single Mitochondrion ◽  
Degree Of Heterogeneity

In order to study mitochondrial turnover at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein developed by Terskikh et al. The Timer protein transitions from green fluorescence to a more stable red conformation as it matures over a span of 48 h. Furthermore, the protein is very stable under physiological conditions, insensitive to variations in ionic strength, and changes in pH between 7.0 and 8.0. Notably, Timer maturation from green to red is significantly slowed in deoxygenated buffer, suggesting that molecular oxygen plays a part in fluorophore maturation. We fused the Timer protein with the mitochondrial signal sequence from the cytochrome c oxidase subunit VIII (COX8) to target the protein to the inner membrane of the mitochondria, and further cloned the protein into a construct with a cardiac-restricted α-myosin heavy chain promoter. This construct was used to create the α-MHC MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice reveals a remarkable degree of heterogeneity in the ratio of red-to- green fluorescence of MitoTimer in cardiac tissue. Furthermore, individual mitochondria within cardiomyocytes display a higher red-to-green fluorescence, implying a block in import of newly synthesized MitoTimer that would be caused by the lack of a high membrane potential, indicative of older, dysfunctional mitochondria. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart.

scholarly journals Direct biologically based biosensing of dynamic physiological function

2001 ◽  
Vol 280 (5) ◽  
pp. H2006-H2010 ◽  
Author(s):  
David J. Christini ◽  
Jeff Walden ◽  
Jay M. Edelberg
Keyword(s):  
Real Time ◽  
Cardiac Tissue ◽  
Physiological Function ◽  
Physiological Activity ◽  
Functional Integration ◽  
Dynamic Regulation ◽  
Excitable Tissue ◽  
Alternative Approach

Dynamic regulation of biological systems requires real-time assessment of relevant physiological needs. Biosensors, which transduce biological actions or reactions into signals amenable to processing, are well suited for such monitoring. Typically, in vivo biosensors approximate physiological function via the measurement of surrogate signals. The alternative approach presented here would be to use biologically based biosensors for the direct measurement of physiological activity via functional integration of relevant governing inputs. We show that an implanted excitable-tissue biosensor (excitable cardiac tissue) can be used as a real-time, integrated bioprocessor to analyze the complex inputs regulating a dynamic physiological variable (heart rate). This approach offers the potential for long-term biologically tuned quantification of endogenous physiological function.

scholarly journals Vectored Immunotherapeutics for Infectious Diseases: Can rAAVs Be The Game Changers for Fighting Transmissible Pathogens?

2021 ◽  
Vol 12 ◽  
Author(s):  
Wei Zhan ◽  
Manish Muhuri ◽  
Phillip W. L. Tai ◽  
Guangping Gao
Keyword(s):  
Infectious Diseases ◽  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
De Novo ◽  
Genomic Variation ◽  
Transduction Efficiency ◽  
Foreign Proteins ◽  
Repeated Dosing

Conventional vaccinations and immunotherapies have encountered major roadblocks in preventing infectious diseases like HIV, influenza, and malaria. These challenges are due to the high genomic variation and immunomodulatory mechanisms inherent to these diseases. Passive transfer of broadly neutralizing antibodies may offer partial protection, but these treatments require repeated dosing. Some recombinant viral vectors, such as those based on lentiviruses and adeno-associated viruses (AAVs), can confer long-term transgene expression in the host after a single dose. Particularly, recombinant (r)AAVs have emerged as favorable vectors, given their high in vivo transduction efficiency, proven clinical efficacy, and low immunogenicity profiles. Hence, rAAVs are being explored to deliver recombinant antibodies to confer immunity against infections or to diminish the severity of disease. When used as a vaccination vector for the delivery of antigens, rAAVs enable de novo synthesis of foreign proteins with the conformation and topology that resemble those of natural pathogens. However, technical hurdles like pre-existing immunity to the rAAV capsid and production of anti-drug antibodies can reduce the efficacy of rAAV-vectored immunotherapies. This review summarizes rAAV-based prophylactic and therapeutic strategies developed against infectious diseases that are currently being tested in pre-clinical and clinical studies. Technical challenges and potential solutions will also be discussed.

A Functional In Vivo RNAi screen Involving Jumonji C Domain Containing Candidates Unravels Kdm5b As a Negative Modulator of Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 385-385
Author(s):  
Sonia Cellot ◽  
Kristin J Hope ◽  
Martin Sauvageau ◽  
Jalila Chagraoui ◽  
Eric Deneault ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Expansion ◽  
Rt Pcr ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Expansion ◽  
Time Point

Abstract Abstract 385 Epigenetic modifications influence chromatin accessibility, impacting on cell fate decisions, such as stem cell self-renewal and differentiation, in both normal and leukemic stem cells (LSC). To investigate the putative role of histone demethylases (HDM) in modulating primary hematopoietic stem cell (HSC) fate, an in vivo functional screen was performed, using an RNAi based strategy, involving 25 members of the Jumonji (JmjC) domain protein family. As a first step, expression profile studies of these gene candidates were undertaken. Transcripts of all these enzymes were detected in isolated HSC populations (frequency 1:2) from fetal liver (n=1) and bone marrow (n=2), except for Hairless. As compared to unsorted bone marrow (BM), stem cells harboured higher expression of Jarid1b (relative-fold enrichment (RQ) of 3.9±1.7), Jmjd2d (RQ3.8±1.9), and Jhdm1b (3.1±1.7). Next, 5shRNA were designed against each of the 25 JmjC containing proteins, and cloned into a retroviral LMP vector encoding GFP to permit tracking of transduced cells in vivo. HSC-enriched CD150+CD48−Lin−cells (∼60 LT-HSC) were infected over 5 days by co-culture with retroviral producer cells in an arrayed 96-well format, with one shRNA per well. Directly after infection, the in vivo reconstituting potential of ¼ of each well was evaluated through duplicate competitive repopulation assays involving the co-transplantation of 1.5 × 105 congenic BM competitor cells into irradiated recipients. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-RT-PCR analysis. Blood reconstitution was evaluated at an early (4wks) and late time point (16–20wks), tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to Luciferase (no effect) and the histone acetyl transferase Myst3 (stem cell loss) were used, as well as Hoxb4 over-expression (stem cell expansion). The primary screen, followed by validation experiments, unravelled one positive (Jhdm1f/Phf8) and two negative (Jarid1b, Hif1an) regulators of HSC activity. The strongest impact was seen with Jarid1b knockdown, and the resulting gain in HSC activity. As a confirmation step, cells were kept in culture for one week, to better contrast an increase in HSC activity, compared to control HSC. After 7 days in vitro, 1/8 equivalents of single well cultures were transplanted into 3 mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against Jarid1b contributed more significantly to host hematopoiesis than sh-RNA Luciferase transduced cells (58±16% vs 26±3% GFP), or Hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 16 week time point and beyond (3 independent experiments). Long-term HSC frequencies were evaluated from these cultures, and found to be 6–10 fold increased in shJari1d1b-cell cultures. In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations confirmed oligoclonality of the stem cell expansion, and HSC pluripotency. There were no cases of leukemic transformation in all of the transplant recipients (n>30). As assessed by Q-RT-PCR, levels of HoxA5, HoxA9, HoxA10 and CxCl5 were increased in day7 sh3Jarib1b-cells (vs ctl), while the levels of the tumor suppressors Cav1, Sash1 and Egr1 were decreased. A more detailed assessment of the HoxA cluster revealed predominant expression of 5' cluster genes in expanding shJarib1b-cells, from HoxA5 to HoxA11, with a concomitant increase in the level of H3K4 tri-methylation, as assessed by ChIP-CHIP. In conclusion, HDM of the JmjC family can modulate HSC activity, both positively and negatively. These data suggest that the H3K4 demethylase Jarid1b (KDM5b) restrains stem cell self-renewal, acting as a co-repressor, possibly via epigenetic regulation of the HoxA gene cluster, among other target genes. This observation could be further exploited as an HSC expansion strategy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Preexisting Neutralizing Antibodies to Adeno-Associated Virus Capsids in Large Animals Other Than Monkeys May Confound In Vivo Gene Therapy Studies

2015 ◽  
Vol 26 (3) ◽  
pp. 103-105 ◽  
Author(s):  
Roberto Calcedo ◽  
Judith Franco ◽  
Qiuyue Qin ◽  
Dean W. Richardson ◽  
Jeffery B. Mason ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Neutralizing Antibodies ◽  
Adeno Associated Virus ◽  
Virus Capsids ◽  
Large Animals ◽  
Therapy Studies

scholarly journals CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX–knockout mice

Blood ◽  
2019 ◽  
Vol 133 (26) ◽  
pp. 2745-2752 ◽  
Author(s):  
Lili Wang ◽  
Yang Yang ◽  
Camilo Ayala Breton ◽  
John White ◽  
Jia Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Partial Hepatectomy ◽  
Neutralizing Antibodies ◽  
Genetic Diseases ◽  
Factor Ix ◽  
Therapeutic Effects ◽  
Vector Genome ◽  
Human Factor Ix

Abstract Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.

Of streaming hepatocytes and meandering sinusoids

1994 ◽  
Vol 13 (9) ◽  
pp. 604-605
Author(s):  
Alan J Paine
Keyword(s):  
Escherichia Coli ◽  
Stem Cells ◽  
Gene Transfer ◽  
Marker Gene ◽  
Nuclear Localisation Signal ◽  
Adult Rat ◽  
Long Term Experiments ◽  
One Year

The fate of normal hepatocytes in adult rat liver was studied after genetic labelling using the Escherichia coli β-galactosidase gene coupled to a nuclear localisation signal. The marker gene was introduced by direct in vivo retroviral-mediated gene transfer into hepatocytes 24 h after partial hepatectomy. Analysis of β-galactosidase expression in the liver at various times after gene transfer revealed that labelled hepatocytes were distributed throughout the entire lobule with a predominance in the periportal and mediolobular regions. Long-term experiments demonstrated that division of hepatocytes did occur as was revealed by the increasing number of β-galactosidase-positive cells in isolated clusters. There was no evidence for the participation of stem cells in this process. Moreover, we found that after more than one year, the pattern of distribution of positive cells within the lobule was not modified. This suggests that hepatocytes do not migrate from the portal space to the perivenous region, as has been previously hypothesised.

scholarly journals Neutralizing Antibodies in COVID-19 Patients and Vaccine Recipients after Two Doses of BNT162b2

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1364
Author(s):  
Julien Favresse ◽  
Constant Gillot ◽  
Laura Di Chiaro ◽  
Christine Eucher ◽  
Marc Elsen ◽  
...  
Keyword(s):  
Dose Regimen ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Neutralization Test ◽  
Virus Neutralization Test ◽  
P Value ◽  
Slow Decay ◽  
Neutralizing Capacity ◽  
Time Point

The evaluation of the neutralizing capacity of anti-SARS-CoV-2 antibodies is important because they represent real protective immunity. In this study we aimed to measure and compare the neutralizing antibodies (NAbs) in COVID-19 patients and in vaccinated individuals. One-hundred and fifty long-term samples from 75 COVID-19 patients were analyzed with a surrogate virus neutralization test (sVNT) and compared to six different SARS-CoV-2 serology assays. The agreement between the sVNT and pseudovirus VNT (pVNT) results was found to be excellent (i.e., 97.2%). The NAb response was also assessed in 90 individuals who had received the complete dose regimen of BNT162b2. In COVID-19 patients, a stronger response was observed in moderate–severe versus mild patients (p-value = 0.0006). A slow decay in NAbs was noted in samples for up to 300 days after diagnosis, especially in moderate–severe patients (r = −0.35, p-value = 0.03). In the vaccinated population, 83.3% of COVID-19-naive individuals had positive NAbs 14 days after the first dose and all were positive 7 days after the second dose, i.e., at day 28. In previously infected individuals, all were already positive for NAbs at day 14. At each time point, a stronger response was observed for previously infected individuals (p-value < 0.05). The NAb response remained stable for up to 56 days in all participants. Vaccinated participants had significantly higher NAb titers compared to COVID patients. In previously infected vaccine recipients, one dose might be sufficient to generate sufficient neutralizing antibodies.

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