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359. Fibrin-Conjugated Pseudotyped Lentivirus for Cell-Controlled and Spatially Localized Gene Delivery on Microarrayed Surfaces
RNA VIRUS VECTORS I 357.
‘Self’ Marker CD47 on Viral Envelopes
Nisha Sosale,1 Richard K. Tsai,1 Irena Ivanovska,1 Philip W. Zoltick,2 Dennis E. Discher.1 1 Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA; 2Department of Surgery, The Children’s Hospital of Philadelphia, Philadelphia, PA. Viruses that bud from the plasma membrane of host cells are enveloped by host cell membrane lipids and - probably - proteins as well. Among these proteins is CD47, which is a ubiquitous ‘Marker of Self’ that can in principle decrease particle clearance (and destruction) by the monocytic phagocyte system. To examine whether viral presentation of CD47 reduces phagocytosis by macrophages, CD47 was displayed at high levels on HIV 1 derived lentiviral vectors. The human-derived 293T packaging cell line was transduced and a stable line created that expressed CD47-GFP, and then these cells were transfected to produce the lentiviral vectors. We use sucrose ultracentrifugation gradient to separate lentiviruses from cell debris. Preliminary data suggests viral display of CD47 by single particle GFP uorescence. Transduction studies of macrophages - which express the counter receptor for CD47 - and other cell types are ongoing, but we expect decreased transduction only of macrophages. Additional motivation for the effort is based on the fact that immune responses to gene therapy vectors and their transgene products is a major problem in the eld of gene therapy. Understanding whether ‘Self’ Marker CD47 on viral envelopes decreases immune uptake is thus of both basic and applied signicance.
358. Lentiviral Microarrays for High-Throughput and Real-Time Monitoring of Gene Expression Dynamics Jun Tian,1 Stella Allimperti,1 Pedro Lei,1 Stelios T. Andreadis.1 Chemical and Biological Engineering, State University of New York at Buffalo, Amherst, NY.
1
Quantication of gene expression dynamics is limited due to the destructive, expensive and laborious nature of current gene expression proling techniques such as qRT-PCR and cDNA microarrays. Here we developed scalable live-cell microarrays to measure gene expression dynamics in real-time and in a high-throughput manner. To this end, we generated dual-promoter lentiviral vectors that were designed to eliminate promoter interference and provide independent and high level gene expression from two gene cassettes. Each lentivirus harbored a transcriptional regulatory element encoding for destabilized green uorescence protein to monitor cell activation in response to an exogenous signal and a constitutive promoter driving red uorescence protein for internal signal normalization. Lentivirus preparations were immobilized in a microarray format using a robotic spotter to generate the LentiVirus microArray (LVA). Target cells were transduced with immobilized lentivirus and after cytokine treatment transcriptional activity was interrogated in real-time using automated uorescence microscopy. In contrast to standard methods, our experiments provided rich dynamic information over a period of several days. Data normalization by red uorescence intensity eliminated errors due to spot-to-spot variability in transduction efciency or changes in cell proliferation upon cytokine treatment. These results were conrmed by ow cytometry and qRT-PCR and demonstrated that the LVA can monitor gene expression in realtime and high-throughput manner thereby providing a useful tool for deciphering gene regulatory networks of complex biological processes such as inammatory response or stem cell differentiation.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
359. Fibrin-Conjugated Pseudotyped Lentivirus for Cell-Controlled and Spatially Localized Gene Delivery on Microarrayed Surfaces Roshan Padmashali, Pedro Lei, Stelios Andreadis. Chemical and Biological Engineering, State University of New York at Buffalo, Amherst, NY.
Recently, we reported that brin hydrogels can be used for effective pDNA encapsulation and gene delivery. Here we report our recent efforts to immobilize lentivirus in brin gels to achieve gene transfer in a cell-controlled and spatially arranged manner. Transduction efciency on brin gels was comparable to conventional methods but cellular toxicity was signicantly diminished. Gene transfer was strongly dependent on matrix degradation by target cells but a signicant fraction of lentiviral particles diffused out of the gel over time. To eliminate viral diffusion we engineered lentiviral particles that bind covalently to brin during polymerization. To this end, we engineered fusion proteins between the viral envelope glycoprotein (VSV-G), peptide domains that are recognized by factor XIII and protease cleavage sites that are recognized by plasmin. All modied variants exhibited similar transduction efciency with the wild type and bound to brin hydrogels in a factor XIII dose dependent manner. As a result diffusion of virus from the gels decreased dramatically even for brin gels with low brinogen concentration. When the modied lentivirus preparations were spotted in an array format, gene transfer was strictly conned to virus-containing brin spots with no cross-contamination between neighboring sites suggesting that this transduction system may be ideal for generation of lentiviral microarrays for high throughput studies.
360. Improved Transduction Efciency of Human Amniotic Mesenchymal Stem Cells Using Optimized Lentiviral Vectors
F. Pisano,1 P. Danieli,1 E. Cervio,1 C. Ciuffreda,1 M. Roccio,1 G. Viarengo,1 M. Gnecchi.1 1 Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
Background: Amniotic mesenchymal stem cells (A-MSC) are excellent candidates for regenerative medicine since they are multipotent, easy to isolate, expandable and seem to be immunoprivileged. In rodents, stable genetic modication with viral vectors improves A-MSC function. Unfortunately, a single cycle transduction with standard viral vectors does not achieve high efciency in human A-MSC. On the other hand, multiple transduction cycles or antibiotic-based selection methods may alter S139
‘Self’ Marker CD47 on Viral Envelopes
Nisha Sosale,1 Richard K. Tsai,1 Irena Ivanovska,1 Philip W. Zoltick,2 Dennis E. Discher.1 1 Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA; 2Department of Surgery, The Children’s Hospital of Philadelphia, Philadelphia, PA. Viruses that bud from the plasma membrane of host cells are enveloped by host cell membrane lipids and - probably - proteins as well. Among these proteins is CD47, which is a ubiquitous ‘Marker of Self’ that can in principle decrease particle clearance (and destruction) by the monocytic phagocyte system. To examine whether viral presentation of CD47 reduces phagocytosis by macrophages, CD47 was displayed at high levels on HIV 1 derived lentiviral vectors. The human-derived 293T packaging cell line was transduced and a stable line created that expressed CD47-GFP, and then these cells were transfected to produce the lentiviral vectors. We use sucrose ultracentrifugation gradient to separate lentiviruses from cell debris. Preliminary data suggests viral display of CD47 by single particle GFP uorescence. Transduction studies of macrophages - which express the counter receptor for CD47 - and other cell types are ongoing, but we expect decreased transduction only of macrophages. Additional motivation for the effort is based on the fact that immune responses to gene therapy vectors and their transgene products is a major problem in the eld of gene therapy. Understanding whether ‘Self’ Marker CD47 on viral envelopes decreases immune uptake is thus of both basic and applied signicance.
358. Lentiviral Microarrays for High-Throughput and Real-Time Monitoring of Gene Expression Dynamics Jun Tian,1 Stella Allimperti,1 Pedro Lei,1 Stelios T. Andreadis.1 Chemical and Biological Engineering, State University of New York at Buffalo, Amherst, NY.
1
Quantication of gene expression dynamics is limited due to the destructive, expensive and laborious nature of current gene expression proling techniques such as qRT-PCR and cDNA microarrays. Here we developed scalable live-cell microarrays to measure gene expression dynamics in real-time and in a high-throughput manner. To this end, we generated dual-promoter lentiviral vectors that were designed to eliminate promoter interference and provide independent and high level gene expression from two gene cassettes. Each lentivirus harbored a transcriptional regulatory element encoding for destabilized green uorescence protein to monitor cell activation in response to an exogenous signal and a constitutive promoter driving red uorescence protein for internal signal normalization. Lentivirus preparations were immobilized in a microarray format using a robotic spotter to generate the LentiVirus microArray (LVA). Target cells were transduced with immobilized lentivirus and after cytokine treatment transcriptional activity was interrogated in real-time using automated uorescence microscopy. In contrast to standard methods, our experiments provided rich dynamic information over a period of several days. Data normalization by red uorescence intensity eliminated errors due to spot-to-spot variability in transduction efciency or changes in cell proliferation upon cytokine treatment. These results were conrmed by ow cytometry and qRT-PCR and demonstrated that the LVA can monitor gene expression in realtime and high-throughput manner thereby providing a useful tool for deciphering gene regulatory networks of complex biological processes such as inammatory response or stem cell differentiation.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
359. Fibrin-Conjugated Pseudotyped Lentivirus for Cell-Controlled and Spatially Localized Gene Delivery on Microarrayed Surfaces Roshan Padmashali, Pedro Lei, Stelios Andreadis. Chemical and Biological Engineering, State University of New York at Buffalo, Amherst, NY.
Recently, we reported that brin hydrogels can be used for effective pDNA encapsulation and gene delivery. Here we report our recent efforts to immobilize lentivirus in brin gels to achieve gene transfer in a cell-controlled and spatially arranged manner. Transduction efciency on brin gels was comparable to conventional methods but cellular toxicity was signicantly diminished. Gene transfer was strongly dependent on matrix degradation by target cells but a signicant fraction of lentiviral particles diffused out of the gel over time. To eliminate viral diffusion we engineered lentiviral particles that bind covalently to brin during polymerization. To this end, we engineered fusion proteins between the viral envelope glycoprotein (VSV-G), peptide domains that are recognized by factor XIII and protease cleavage sites that are recognized by plasmin. All modied variants exhibited similar transduction efciency with the wild type and bound to brin hydrogels in a factor XIII dose dependent manner. As a result diffusion of virus from the gels decreased dramatically even for brin gels with low brinogen concentration. When the modied lentivirus preparations were spotted in an array format, gene transfer was strictly conned to virus-containing brin spots with no cross-contamination between neighboring sites suggesting that this transduction system may be ideal for generation of lentiviral microarrays for high throughput studies.
360. Improved Transduction Efciency of Human Amniotic Mesenchymal Stem Cells Using Optimized Lentiviral Vectors
F. Pisano,1 P. Danieli,1 E. Cervio,1 C. Ciuffreda,1 M. Roccio,1 G. Viarengo,1 M. Gnecchi.1 1 Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
Background: Amniotic mesenchymal stem cells (A-MSC) are excellent candidates for regenerative medicine since they are multipotent, easy to isolate, expandable and seem to be immunoprivileged. In rodents, stable genetic modication with viral vectors improves A-MSC function. Unfortunately, a single cycle transduction with standard viral vectors does not achieve high efciency in human A-MSC. On the other hand, multiple transduction cycles or antibiotic-based selection methods may alter S139