- Email: [email protected]
808. The Two Phases of Measles Virus Cell Entry through SLAM: Hemagglutinin Residues Necessary for Primary Binding or Membrane Fusion
and Fv-I interact with the incoming retroviral core (capsid) and are expected to act before and after viral DNA synthesis, respectively, these studies reveal that PMT is indeed mediated by retroviral particles. Strikingly, PMTwas downregulated in target cells expressing a siRNA against the incoming viral mRNA, revealing that mRNA and not protein transfer is responsible for transient cell modification. Our data thus suggest that retrovirally delivered mRNA serves as an immediate trans lation template if not being reverse transcribed. We conclude that RT-deficient retroviral vectors hold great promise for applications in which low and transient expression of proteins achieves striking biological effects,
806. Psuedotyping Lentiviral Vector Particles for the Envelope Protein from Feline Leukemia Virus Type C (FLVC) Matthew Wielgosz,' Phillip Hargrove: Steven Kepes,' John Gray, I Derek Persons, I Arthur Nienhuis. ' IDivision 0/Experimental Hematology, St. Jude Children s Research Hospital, Memphis, TN. The ability to genetically modify hematopoietic stem cells and achieve regulated gene expression in specific lineages will provide many therapeutic opportunities for inherited blood diseases. We have developed a lentiviral vector system based on simian immunodeficiency virus (SIV) with the goal of optimizing gene transfer efficicney in a rhesus, non-human primate model (Blood 103:4062-9, 2004). An average of 18 ± 8% genetically modified granulocytes were present 4-6 months following transplantation of transduced, mobilized peripheral blood stem cells. These studies were performed with vector particles pseudotyped with the oncoretroviral amphotrophic (AMPHO) envelope protein . Recent studies have shown the mRNA for the receptor for the FLVC envelope protein is present in higher concentrations in the most primitive hematopoietic cells (Blood 106:51-8, 2005) in contrast to the mRNA for the receptor for theAMPHO envelope protein which is present at lower concentrations in the earliest cells. Accordingly, we have devised strategies to pseudotype lentiviral vector particles with the FLVC envelope protein to test the hypothesis that such particles will be more efficient at transducing repopulating stem cells. We generated four dilTerent FLVC envelope constructs: I) FLVC, 2) FLVC ehimcra (fusion of the FLVC surface unit (SU), and the AMPHO transmembrane (TM) envelope proteins), 3) SA FLVC (containing the AMPHO splice acceptor (SA) in place ofthe FLVC SA), and 4) SA FLVC Chimera (containing theAMPHO SAand TM, in place of the corresponding FLVC SA and TM), in the context of the pCAG4 envelope expression construct (Molecular Therapy 5:242-51.2002), and tested their abilities to pseudotype SIV particles. Transfeetion of these viral envelope constructs into 293T eells, in the presence ofthe requisite SIV packaging plasm ids and SIV transfer vector generated conditioned media with low titers of infectious vector particles on Hel.acellsj-ctxln' IUlml). However, when the FLVCgagpol(FGP) plasmid was transfected with the previously mentioned plasm ids, SIV(FLVC) titers consistently increased 2-fold, i.e. 1-2x IO~ IUlml versus 0.4-1 x I O~ IUlml. In light of these results, we constructed the FLVC R-Less envelope construct, hypothesizing that inefficient cleavage ofthe R-peptide from the cytoplasmic tail ofthe FLVCTM envelope protein , limited its ability to mediate virus-target membrane fusion. The use of the FLVC R-Less envelope consistently yielded SIV(FLVC) titers of2x IOs IUlml, and concentrated titers of 1-2x107 IUlml (via ultracentrifugation). In contraslto SlY, the FLVC R-Less envelope did not increase HIV (FLVC) titers compared to the R-peptide containing FLVC envelope, (6 versus 7xlO~ IUlml, respectively), suggesting that the assembly of HIV pseudotyped particles may dilTer from SIV. Using concentrated vector preparations with titers of 107, we have been able to transduce 30-35% of the clonogenie progenitors in rhesus CD34+ cell populations with S3IO
FLVC pseudotyped vector particles. We are now positioned to test the hypothesis that such particles will efficiently transduce primitive repopulating cells in the rhesus model.
807. Engineering the Tropism of VSVGPseudotyped Retroviral and Lentiviral Vectors Via the Insertion of RGD Targeting Peptides Kwang-il Lim, Julie Yu, David Schaffer, 'Chemical Engineering. University ofCalifornia at Berkeley; Berkeley, CA. Pseudotyping retroviral and lentiviral vectors with the vesicular stomatitis virus glycoprotein envelope (VSVG) has not only increased their stability, allowing enhanced purification processes of harsh conditions, but also extended their infectivity to most cell types. Although such tropism extension greatly facilitates the usc of retrovira I vectors for delivering therapeutic genes to various cell populations in vitro, the associated promiscuity limits their ability to deliver genes to specific target cells in vivo. To test whether the broad tropism ofVSVG-pseudotyped retroviral vectors can be restricted to specific target receptors , we genetically inserted six or fourteen amino acid long RGD and RGE peptide motifs into one of several permissive peptide insertion sites within VSVG that we have recently identified (Yu and SchalTer, J. Virology, 2006). The ROD motifs can interact with several integrin cell surface adhesion receptors, whereas the negative control RGE peptides cannot bind integrins. Importantly, these peptide insertions into our previously identified permissive site near the N-terminus ofVSVG did not cause a significant decrease in viral packaging efficiency. More interestingly, a single amino acid dilTerence between ROE to ROD in the targeting peptides led to substantially increased viral infectivities of up to 10fold for 293T (embryonic kidney), TF-I (leukemia-derived progenitor), M07E (leukemia-derived progenitor) and 1316FI0 (melanoma) cells, which each express distinct sets of RGD-binding integrin receptors as assessed by RT-PCR. Furthermore, overexpression of some integrin heterodimers in 293T cells significantly increased their infection by lentivai vectors pseudotyped with VSVG-RGD proteins. These results suggest that pseudotyping retroviral and lentiviral vectors with VSVO variants carrying targeting peptides in the right location can achieve selective gene delivery via binding to novel cellular receptors.
808. The Two Phases of Measles Virus Cell Entry through SLAM: Hemagglutinin Residues Necessary for Primary Binding or Membrane Fusion Chanakha K. Navaratnarajah,' Sompong Vongpunsawad,' Numan
Oezgun.' Rachel Trehin,' Thilo Stehle,' Werner Braun,' Roberto Cattaneo.' 'Molecular Medicine Program and Virologyand Gene Therapy Track, Mayo Clinic College ofMedicine, Rochester, MN; lSealy Center/or Structural Biology, University ofTexas Medical Branch, Galveston, TX; J Interfakultares lnstitut /iir Biochemic, University ofTiibingen. Tiibingen, Germany.
Measles virus (MV) is an enveloped negative strand RNA virus of the family Paramyxoviridae and genus Morbillivirus. MV is the first recombinant RNA virus in phase I clinical trials of oncolysis, and receptor-retargeted MV have enhanced oncolytic specificity and efficacy in pre-clinical trials. All strains of MV recognize the signaling lymphocyte-activation molecule (SLAM; also known as CD 150); in addition, the live attenuated Edmonston strain enters cells via the ubiquitous regulator of complement activation CD46. Previously we identified a cluster of four mutants in beta-sheet 5 of the MV attachment protein hemagglutinin (H) that is necessary for SLAM-dependent fusion function. In order to identify addiMolecular Therapy Volume 15, Supplemen t I. ,\br 2007 Co pyright © Th e American Society o f G ene Thcr.Lp)·
tional SLAM-relevant residues , amino acids constituting potential interacting surfaces of the H protein as predicted by InterProSurf computer analysis were subjected to mutagenesis. InterProSurf predicts potential interacting residues based on the solvent accessible surface area in our 3D model of MV H. Based on the analysis of these mutations we have identified an additional residue isoleucine 194, located on the top of beta-sheet I, as being necessary for SLAM-dependent fusion. We then produced soluble eetodomains of these H mutants and measured their association and dissociation constants to both receptors by surface plasmon resonance. Substituting isoleucine 194 with serine weakens SLAM-binding by 50 fold while having minimal effect on the CD46 interaction. In contrast, the bs5-quartet (quartet of mutants in beta-sheet 5) has only minor effects on attachment to SLAM and CD46, suggesting that these residues arc not directly involved in SLAM binding, but playa role in SLAM-induced fusion. These data indicate that 1194 is required for efficient SLAM binding. By producing a series of I194 amino acid substitutions with scaled affinity we expect to determine the affinity threshold still allowing efficient entry. Taken together, the data indicate that two phases of SLAM -dependent entry exist: primary binding , during which II 94 on beta-sheet I contacts SLAM, and a subsequent step involving the quartet of residues on beta-sheet 5. Attachment protein mutants of both classes should be considered when designing SLAM-blind MV for oncolysis.
809. Two Viruses: Similar Fibers, Similar Receptors, Different Destinies Katherine J. D. A. Excoffon ,' Nicholas D. Gansemer,' J. Denise Wetzel,' Kristen M. Guglielmi,' Jacquelyn A. Campbell,' Joseph Zahner,' Terence S. Dermody.' 'Internal Medicine. University 0/1011'0, Iowa City, IA; "Eiizabeth B. Lamb Center/or Pediatric Research. Vanderbilt University School a/Medicine. Nashville, TN. Reovirus and adenovirus are members of different virus families and have RNA or DNA genomes, respectively. However, they share striking similarities, including similar fiber-like attachment proteins , sigma I and fiber, similar receptors , JAM-A and CAR, and potential usc in oncolytic virus therapy. To determine the efficiency and potential for reovirus as a pulmonary gene-transfer vehicle, we investigated the biology of reovirus infection using primary cultures of human airway epithelia grown at the air-liquid interface. We hypothesized that if JAM-A was localized to the basolateral membrane of human airway epithelia, reovirus would preferentially infect the basolateral surface, similar to adenovirus. We further hypothesized that a sialic acid-binding reovirus strain might overcome this limitation and efficiently infect from the apical surface. human airway epithelia were infected either apically or basolaterally with sialic acid-binding (T3SA+) or non-sialic acid-binding (T3SA-) strains and evaluated for JAM-A localization. junctional integrity, viral replication, and viral egress. We found that JAM-A is localized to the basolateral membrane in human airway epithelia and, concordantly, reovirus infection was more efficient from the basolateral surface. Infection by T3SA+ was reduced in comparison to infection by T3SA-. Pretreatment of human airway epithelia with neuraminidase enhanced T3SA+ infection, suggesting that sialic acid binding inhibits infection from either the apieal or basolateral membrane. In contrast to adenovirus, which replicates and spreads laterally, disrupting junctional integrity resulting in apical escape, reovirus does not disrupt junctional integrity, and progeny virus is shed directly into the apical compartment. Thus, reovirus infection in airway epithelia is distinct from adenovirus infection. Reovirus does not efficiently infect airway epithelia from the apical surface and uses a pathway of pulmonary egress that does not alter junctional integrity. Molecular Therapy Volume 15.Supplement I. ,\ br 2007 Copyright© The Arncric...an Society of (jl:nc 1111:r:1PY
810. Increasing Incorporation of HN Enhances Infectivity of HPIV3-Pseudotyped Lentiviruses Cindy Jung,' Joseph M. Le Doux.' 'The Wallace H. Coulter Department 0/Biomedical Engineering. Georgia Institute of Technology and Emory University, Atlanta. GA. We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer (3.5 x 102 cfu/mL on 293T cells). We examined whether titers were low due to inadequate expression by the virus producer cells of HN and F, or because too few HN and F were incorporated into the virus particles . As a first step to determine why titers were low, we compared the mRNA and protein expression levels of HN and F in transfeeted cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels ofI-IN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8 and 1.3-fold less, respectively) than cells infected with wild-type HPIV3. To increase expression ofHN in transfected cells, we codon optimized HN and used it to transfeet lcntivirus producer cells. Cell surface expression ofHN, as well as the amount ofHN incorporated into virus particles, increased 2 to 3-fold. Virus titers increased 1.2 to 6A-fold, and the transduction efficiency of polarized MOCK cells via their apical surfaces increased lA-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that I-IPIV3 pseudotyped viruses contained significantly fewer envelope proteins than lentiviruses pseudotyped with the amphotropie envelope protein. Our findings suggest that the titers of I-IPIV3 pseudotyped lentiviruses arc low, not because the producer cells express inadequate levels of the envelope prote ins, but because the lentiviruses incorporate too few of these proteins to be able to efficiently transduce cells.
811. Titers of HIV-Based Vectors Encoding shRNAs Are Reduced by a Dicer-Dependent Mechanism Anantha Lakshmi Poluri, Richard E. Sutton. JDepartment a/Molecular Virology and Microbiology; Baylor College a/Medicine. Houston. Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful for deciphering gene function and are being considered for therapeutic knock-down oftarget genes in man. We constructed I-IIV-based vectors encoding shRNA against I-IIV co-receptor CCR5. Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers compared to the empty vector. Co-transfection of expression plasm ids encoding CCR5 in the producer cells yielded a 10-fold increase in viral titer, indicating that CCR5 mRNA rather than HIV vector mRNA could be targeted by CCR5 shRNA. Addition to the producers of Nodamura virus B2 protein or Adenovirus VAl RNA, inhibitors of'the Dicer-dependent siRNA pathway, increased vector titer to almost empty vector levels. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative RT-PCR suggested that the effects were in part due to reduction of vector RNA in the producers. Similar results were observed with a rctroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism , which to a large extent can be reversed by inhibition ofthat pathway. This may have important implications for large-scale production ofRNA vectors encoding shRNAs.
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806. Psuedotyping Lentiviral Vector Particles for the Envelope Protein from Feline Leukemia Virus Type C (FLVC) Matthew Wielgosz,' Phillip Hargrove: Steven Kepes,' John Gray, I Derek Persons, I Arthur Nienhuis. ' IDivision 0/Experimental Hematology, St. Jude Children s Research Hospital, Memphis, TN. The ability to genetically modify hematopoietic stem cells and achieve regulated gene expression in specific lineages will provide many therapeutic opportunities for inherited blood diseases. We have developed a lentiviral vector system based on simian immunodeficiency virus (SIV) with the goal of optimizing gene transfer efficicney in a rhesus, non-human primate model (Blood 103:4062-9, 2004). An average of 18 ± 8% genetically modified granulocytes were present 4-6 months following transplantation of transduced, mobilized peripheral blood stem cells. These studies were performed with vector particles pseudotyped with the oncoretroviral amphotrophic (AMPHO) envelope protein . Recent studies have shown the mRNA for the receptor for the FLVC envelope protein is present in higher concentrations in the most primitive hematopoietic cells (Blood 106:51-8, 2005) in contrast to the mRNA for the receptor for theAMPHO envelope protein which is present at lower concentrations in the earliest cells. Accordingly, we have devised strategies to pseudotype lentiviral vector particles with the FLVC envelope protein to test the hypothesis that such particles will be more efficient at transducing repopulating stem cells. We generated four dilTerent FLVC envelope constructs: I) FLVC, 2) FLVC ehimcra (fusion of the FLVC surface unit (SU), and the AMPHO transmembrane (TM) envelope proteins), 3) SA FLVC (containing the AMPHO splice acceptor (SA) in place ofthe FLVC SA), and 4) SA FLVC Chimera (containing theAMPHO SAand TM, in place of the corresponding FLVC SA and TM), in the context of the pCAG4 envelope expression construct (Molecular Therapy 5:242-51.2002), and tested their abilities to pseudotype SIV particles. Transfeetion of these viral envelope constructs into 293T eells, in the presence ofthe requisite SIV packaging plasm ids and SIV transfer vector generated conditioned media with low titers of infectious vector particles on Hel.acellsj-ctxln' IUlml). However, when the FLVCgagpol(FGP) plasmid was transfected with the previously mentioned plasm ids, SIV(FLVC) titers consistently increased 2-fold, i.e. 1-2x IO~ IUlml versus 0.4-1 x I O~ IUlml. In light of these results, we constructed the FLVC R-Less envelope construct, hypothesizing that inefficient cleavage ofthe R-peptide from the cytoplasmic tail ofthe FLVCTM envelope protein , limited its ability to mediate virus-target membrane fusion. The use of the FLVC R-Less envelope consistently yielded SIV(FLVC) titers of2x IOs IUlml, and concentrated titers of 1-2x107 IUlml (via ultracentrifugation). In contraslto SlY, the FLVC R-Less envelope did not increase HIV (FLVC) titers compared to the R-peptide containing FLVC envelope, (6 versus 7xlO~ IUlml, respectively), suggesting that the assembly of HIV pseudotyped particles may dilTer from SIV. Using concentrated vector preparations with titers of 107, we have been able to transduce 30-35% of the clonogenie progenitors in rhesus CD34+ cell populations with S3IO
FLVC pseudotyped vector particles. We are now positioned to test the hypothesis that such particles will efficiently transduce primitive repopulating cells in the rhesus model.
807. Engineering the Tropism of VSVGPseudotyped Retroviral and Lentiviral Vectors Via the Insertion of RGD Targeting Peptides Kwang-il Lim, Julie Yu, David Schaffer, 'Chemical Engineering. University ofCalifornia at Berkeley; Berkeley, CA. Pseudotyping retroviral and lentiviral vectors with the vesicular stomatitis virus glycoprotein envelope (VSVG) has not only increased their stability, allowing enhanced purification processes of harsh conditions, but also extended their infectivity to most cell types. Although such tropism extension greatly facilitates the usc of retrovira I vectors for delivering therapeutic genes to various cell populations in vitro, the associated promiscuity limits their ability to deliver genes to specific target cells in vivo. To test whether the broad tropism ofVSVG-pseudotyped retroviral vectors can be restricted to specific target receptors , we genetically inserted six or fourteen amino acid long RGD and RGE peptide motifs into one of several permissive peptide insertion sites within VSVG that we have recently identified (Yu and SchalTer, J. Virology, 2006). The ROD motifs can interact with several integrin cell surface adhesion receptors, whereas the negative control RGE peptides cannot bind integrins. Importantly, these peptide insertions into our previously identified permissive site near the N-terminus ofVSVG did not cause a significant decrease in viral packaging efficiency. More interestingly, a single amino acid dilTerence between ROE to ROD in the targeting peptides led to substantially increased viral infectivities of up to 10fold for 293T (embryonic kidney), TF-I (leukemia-derived progenitor), M07E (leukemia-derived progenitor) and 1316FI0 (melanoma) cells, which each express distinct sets of RGD-binding integrin receptors as assessed by RT-PCR. Furthermore, overexpression of some integrin heterodimers in 293T cells significantly increased their infection by lentivai vectors pseudotyped with VSVG-RGD proteins. These results suggest that pseudotyping retroviral and lentiviral vectors with VSVO variants carrying targeting peptides in the right location can achieve selective gene delivery via binding to novel cellular receptors.
808. The Two Phases of Measles Virus Cell Entry through SLAM: Hemagglutinin Residues Necessary for Primary Binding or Membrane Fusion Chanakha K. Navaratnarajah,' Sompong Vongpunsawad,' Numan
Oezgun.' Rachel Trehin,' Thilo Stehle,' Werner Braun,' Roberto Cattaneo.' 'Molecular Medicine Program and Virologyand Gene Therapy Track, Mayo Clinic College ofMedicine, Rochester, MN; lSealy Center/or Structural Biology, University ofTexas Medical Branch, Galveston, TX; J Interfakultares lnstitut /iir Biochemic, University ofTiibingen. Tiibingen, Germany.
Measles virus (MV) is an enveloped negative strand RNA virus of the family Paramyxoviridae and genus Morbillivirus. MV is the first recombinant RNA virus in phase I clinical trials of oncolysis, and receptor-retargeted MV have enhanced oncolytic specificity and efficacy in pre-clinical trials. All strains of MV recognize the signaling lymphocyte-activation molecule (SLAM; also known as CD 150); in addition, the live attenuated Edmonston strain enters cells via the ubiquitous regulator of complement activation CD46. Previously we identified a cluster of four mutants in beta-sheet 5 of the MV attachment protein hemagglutinin (H) that is necessary for SLAM-dependent fusion function. In order to identify addiMolecular Therapy Volume 15, Supplemen t I. ,\br 2007 Co pyright © Th e American Society o f G ene Thcr.Lp)·
tional SLAM-relevant residues , amino acids constituting potential interacting surfaces of the H protein as predicted by InterProSurf computer analysis were subjected to mutagenesis. InterProSurf predicts potential interacting residues based on the solvent accessible surface area in our 3D model of MV H. Based on the analysis of these mutations we have identified an additional residue isoleucine 194, located on the top of beta-sheet I, as being necessary for SLAM-dependent fusion. We then produced soluble eetodomains of these H mutants and measured their association and dissociation constants to both receptors by surface plasmon resonance. Substituting isoleucine 194 with serine weakens SLAM-binding by 50 fold while having minimal effect on the CD46 interaction. In contrast, the bs5-quartet (quartet of mutants in beta-sheet 5) has only minor effects on attachment to SLAM and CD46, suggesting that these residues arc not directly involved in SLAM binding, but playa role in SLAM-induced fusion. These data indicate that 1194 is required for efficient SLAM binding. By producing a series of I194 amino acid substitutions with scaled affinity we expect to determine the affinity threshold still allowing efficient entry. Taken together, the data indicate that two phases of SLAM -dependent entry exist: primary binding , during which II 94 on beta-sheet I contacts SLAM, and a subsequent step involving the quartet of residues on beta-sheet 5. Attachment protein mutants of both classes should be considered when designing SLAM-blind MV for oncolysis.
809. Two Viruses: Similar Fibers, Similar Receptors, Different Destinies Katherine J. D. A. Excoffon ,' Nicholas D. Gansemer,' J. Denise Wetzel,' Kristen M. Guglielmi,' Jacquelyn A. Campbell,' Joseph Zahner,' Terence S. Dermody.' 'Internal Medicine. University 0/1011'0, Iowa City, IA; "Eiizabeth B. Lamb Center/or Pediatric Research. Vanderbilt University School a/Medicine. Nashville, TN. Reovirus and adenovirus are members of different virus families and have RNA or DNA genomes, respectively. However, they share striking similarities, including similar fiber-like attachment proteins , sigma I and fiber, similar receptors , JAM-A and CAR, and potential usc in oncolytic virus therapy. To determine the efficiency and potential for reovirus as a pulmonary gene-transfer vehicle, we investigated the biology of reovirus infection using primary cultures of human airway epithelia grown at the air-liquid interface. We hypothesized that if JAM-A was localized to the basolateral membrane of human airway epithelia, reovirus would preferentially infect the basolateral surface, similar to adenovirus. We further hypothesized that a sialic acid-binding reovirus strain might overcome this limitation and efficiently infect from the apical surface. human airway epithelia were infected either apically or basolaterally with sialic acid-binding (T3SA+) or non-sialic acid-binding (T3SA-) strains and evaluated for JAM-A localization. junctional integrity, viral replication, and viral egress. We found that JAM-A is localized to the basolateral membrane in human airway epithelia and, concordantly, reovirus infection was more efficient from the basolateral surface. Infection by T3SA+ was reduced in comparison to infection by T3SA-. Pretreatment of human airway epithelia with neuraminidase enhanced T3SA+ infection, suggesting that sialic acid binding inhibits infection from either the apieal or basolateral membrane. In contrast to adenovirus, which replicates and spreads laterally, disrupting junctional integrity resulting in apical escape, reovirus does not disrupt junctional integrity, and progeny virus is shed directly into the apical compartment. Thus, reovirus infection in airway epithelia is distinct from adenovirus infection. Reovirus does not efficiently infect airway epithelia from the apical surface and uses a pathway of pulmonary egress that does not alter junctional integrity. Molecular Therapy Volume 15.Supplement I. ,\ br 2007 Copyright© The Arncric...an Society of (jl:nc 1111:r:1PY
810. Increasing Incorporation of HN Enhances Infectivity of HPIV3-Pseudotyped Lentiviruses Cindy Jung,' Joseph M. Le Doux.' 'The Wallace H. Coulter Department 0/Biomedical Engineering. Georgia Institute of Technology and Emory University, Atlanta. GA. We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer (3.5 x 102 cfu/mL on 293T cells). We examined whether titers were low due to inadequate expression by the virus producer cells of HN and F, or because too few HN and F were incorporated into the virus particles . As a first step to determine why titers were low, we compared the mRNA and protein expression levels of HN and F in transfeeted cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels ofI-IN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8 and 1.3-fold less, respectively) than cells infected with wild-type HPIV3. To increase expression ofHN in transfected cells, we codon optimized HN and used it to transfeet lcntivirus producer cells. Cell surface expression ofHN, as well as the amount ofHN incorporated into virus particles, increased 2 to 3-fold. Virus titers increased 1.2 to 6A-fold, and the transduction efficiency of polarized MOCK cells via their apical surfaces increased lA-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that I-IPIV3 pseudotyped viruses contained significantly fewer envelope proteins than lentiviruses pseudotyped with the amphotropie envelope protein. Our findings suggest that the titers of I-IPIV3 pseudotyped lentiviruses arc low, not because the producer cells express inadequate levels of the envelope prote ins, but because the lentiviruses incorporate too few of these proteins to be able to efficiently transduce cells.
811. Titers of HIV-Based Vectors Encoding shRNAs Are Reduced by a Dicer-Dependent Mechanism Anantha Lakshmi Poluri, Richard E. Sutton. JDepartment a/Molecular Virology and Microbiology; Baylor College a/Medicine. Houston. Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful for deciphering gene function and are being considered for therapeutic knock-down oftarget genes in man. We constructed I-IIV-based vectors encoding shRNA against I-IIV co-receptor CCR5. Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers compared to the empty vector. Co-transfection of expression plasm ids encoding CCR5 in the producer cells yielded a 10-fold increase in viral titer, indicating that CCR5 mRNA rather than HIV vector mRNA could be targeted by CCR5 shRNA. Addition to the producers of Nodamura virus B2 protein or Adenovirus VAl RNA, inhibitors of'the Dicer-dependent siRNA pathway, increased vector titer to almost empty vector levels. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative RT-PCR suggested that the effects were in part due to reduction of vector RNA in the producers. Similar results were observed with a rctroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism , which to a large extent can be reversed by inhibition ofthat pathway. This may have important implications for large-scale production ofRNA vectors encoding shRNAs.
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