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476. Alpha-1 Antitrypsin Delivered to T Cells by rSV40 Vectors Inhibits HIV Morphogenesis by Inhibiting both Cellular Serine Proteases and HIV-1 Protease
GENE THERAPIES AND VACCINES AGAINST INFECTIOUS DISEASES 476. Alpha-1 Antitrypsin Delivered to T Cells by rSV40 Vectors Inhibits HIV Morphogenesis by Inhibiting both Cellular Serine Proteases and HIV-1 Protease Pierre Cordelier,1 David S. Strayer.1 1 Pathology, Jefferson Medical College, Philadelphia, PA, United States. Background: Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120; HIV protease (PR), an aspartyl protease, cleaves p55 Gag to p24, etc., in budding virions. α1antitrypsin (α1AT) is cleaved by serine proteases, causing a conformational change in α1AT that sequesters and so inactivates the protease. α1AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that α1AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, α1AT bound and was itself cleaved by the proteases whose activities were blocked. Methods: SV(AT), a recombinant SV40-derived vector, carries human α1AT cDNA, driven by the CMV promoter. Human T lymphocyte cell lines and primary human blood lymphocytes (PBL) were transduced with SV(AT). No toxicity was detected. HIV replication was measured as supernatant p24 antigen by ELISA in unselected SV(AT)-transduced T cell lines and PBL that had been challenged with HIV-1NL4-3. Immunoprecipitates of these cells were made using antibodies to α1AT, PR and furin and were analyzed by Western blotting for coprecipitated furin, PR and α1AT. Interactions among purified α1AT, PR, and p55 were tested in tube, and results visualized by SDS-PAGE. Results: SV(AT) transduction inhibited processing of both gp120 and p55Gag into their substituents, resulting in decreased incorporation of the mature forms of these precursors into HIV virions. Consequently, SV(AT) completely protected both T cell lines and PBL from challenge, even with very high doses of HIV. α1AT delivered by SV(AT) coprecipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, α1AT and HIV p55Gag were both detected in resulting HIV virions, but p24 was not. HIV1 virions from control-transduced, HIV-1 challenged, cells showed no p55, but only its cleavage products, p24, p17, etc.. α1AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis showed that α1AT directly bound HIV-1 PR, and was cleaved by it. Conclusions: α1AT strongly inhibits HIV replication at two key steps: it blocks the processing of gp160 in the cell and p55 in HIV virions. These events occur through separate α1AT interactions with furin, and perhaps other serine proteases, and HIV-1 PR. The latter represents a novel pattern of α1AT inhibition of protease activity. rSV40delivered α1AT inhibited cellular enzymes that play an obligatory role in HIV infectivity is unlikely to be circumvented by mutations in HIV genes. The simultaneous inhibition of two different steps in HIV morphogenesis both increases α1AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.
477. Potent Inhibitors of Hepatitis C Virus Internal Ribosome Entry Site Translation in Mice Anton P. McCaffrey,1 Leonard Muese,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA, United States. Hepatitis C virus (HCV) is a positive strand RNA virus that infects 170 million people worldwide and is the most common reason for liver transplantation in the western world. Current treatments for HCV (interferon or interferon and ribavirin) are not very effective and result in elimination of the virus in only 20% - 40% of patients. New treatments for HCV are urgently needed. Translational initiation of the HCV polypeptide occurs in a cap independent fashion at an internal ribosome entry site (IRES). The conserved sequence of this domain makes it an attractive target for therapeutic intervention. A mouse model that enables in vivo measurements of translational initiation by the HCV internal ribosome entry site (IRES) was created by hydrodynamics-based transfection of HCV IRES luciferase fusion plasmids. Translation of the fusion was monitored by quantitative whole body imaging of in vivo luciferase activity. Morpholino antisense oligonucleotides were codelivered with reporter plasmids and were evaluated for their ability to inhibit translational initiation by the HCV IRES. A 25 mer oligonucleotide inhibited HCV IRES translation by more than 97 %. A 25 mer inverted
S156
sequence showed no inhibition, however a 25 mer oligonuclotide containing 4 mismatched bases inhibited as well as the perfect complement. To obtain specificity while maintaining potency, a 20 mer oligonucleotide was tested. This oligonucleotide also inhibited HCV IRES translation by more than 97 % for six days. This inhibition was dose dependent. Importantly, a 20 mer with 4 mismatched bases did not show any inhibition. To further evaluate the specificity of the 20 mer oligonucleotide, studies were carried out with reporter plasmids expressing a luciferase gene lacking an IRES or a luciferase gene whose expression was controlled by the encephalomyocarditis virus IRES. Reassuringly, no inhibition of these reporters was observed. In conclusion, we have demonstrated that a 20 mer morpholino oligonucleotide is a potent and specific inhibitor of HCV IRES mediated translation in mice. These studies should aid in the development of therapeutics for the treatment of HCV.
GENE TRANSFER FOR HEMOPHILIA AND OTHER INHERITED DISEASES 478. Development of an AAV-2 Vector for the Treatment of Hemophilia B Thomas C. Harding,1 Kathyrn E. Koprivnikar,1 Melinda J. VanRoey,1 Satya Yendluri,1 Jeff Waugh,1 Brian Donahue.1 1 Research, Cell Genesys, Inc., Foster City, CA, United States. Hemophilia B, the deficiency in the clotting factor IX (FIX), is an Xlinked recessive disorder that occurs in about one in 25,000 males in the U.S. Although the use of plasma-derived or recombinant FIX has greatly extended the life span of these patients, they still remain susceptible to a number of pathologies of the disease including retinopathies, joint bleeding leading to joint dysfunction, vascular occlusive disease, and internal bleeding events that can be life threatening. The use of replication deficient AAV vectors to deliver the human FIX gene offers the opportunity to achieve stable therapeutic levels of FIX in hemophilia patients and the potential to avoid many of these pathologies. We have previously demonstrated the stable expression of human factor IX (hFIX) protein in mice and canine factor IX (cFIX) protein in hemophilic B dogs following gene transfer with a rAAV encoding vector, however, we wished to further optimize the vector transgene cassette used in these studies for tissue specificity and strength of expression. First, several liver-specific promoters were explored and tested. Our rationale was that liver-specific FIX expression would limit gene expression to cells that normally express Factor IX and would help reduce the potential of inducing an anti-hFIX immune response. In addition, the construct was further optimized for expression by the inclusion of additional regulatory elements. Vectors were tested both in cell culture and in vivo for their effect on transgene expression, in comparison to previous generations of AAV-hFIX construct. Results demonstrated that constructs which contained both the b-globin intron and the woodchuck post-transcriptional regulatory element (WPRE) in combination with a liver specific promoter resulted in the strongest expression in vivo, with serum hFIX levels being 5 fold greater than that achieved for an MFG promoter construct. These levels remained stable for over 78 weeks. Although portal vein administration of vector appears to be an effective route of administration to achieve AAV transduction of the liver, it is rather invasive. We therefore also examined alternative delivery routes that maybe more appropriate in the context of hemophilia such as intra-venous (I.V.) and direct liver administration. Results indicate that while the efficiency of gene transfer with I.V. administration of this vector is slightly lower than portal vein administration (70-80% of portal vein values), this route of delivery achieves therapeutic levels of circulating hFIX. Biodistribution was determined by analyses of tissue for the presence of viral vector using real time PCR and demonstrated the predominant organ of transduction was the liver, regardless of administration route. Further studies utilizing this vector will also be presented.
Molecular Therapy Vol. 5, No. 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
477. Potent Inhibitors of Hepatitis C Virus Internal Ribosome Entry Site Translation in Mice Anton P. McCaffrey,1 Leonard Muese,1 Mark A. Kay.1 1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA, United States. Hepatitis C virus (HCV) is a positive strand RNA virus that infects 170 million people worldwide and is the most common reason for liver transplantation in the western world. Current treatments for HCV (interferon or interferon and ribavirin) are not very effective and result in elimination of the virus in only 20% - 40% of patients. New treatments for HCV are urgently needed. Translational initiation of the HCV polypeptide occurs in a cap independent fashion at an internal ribosome entry site (IRES). The conserved sequence of this domain makes it an attractive target for therapeutic intervention. A mouse model that enables in vivo measurements of translational initiation by the HCV internal ribosome entry site (IRES) was created by hydrodynamics-based transfection of HCV IRES luciferase fusion plasmids. Translation of the fusion was monitored by quantitative whole body imaging of in vivo luciferase activity. Morpholino antisense oligonucleotides were codelivered with reporter plasmids and were evaluated for their ability to inhibit translational initiation by the HCV IRES. A 25 mer oligonucleotide inhibited HCV IRES translation by more than 97 %. A 25 mer inverted
S156
sequence showed no inhibition, however a 25 mer oligonuclotide containing 4 mismatched bases inhibited as well as the perfect complement. To obtain specificity while maintaining potency, a 20 mer oligonucleotide was tested. This oligonucleotide also inhibited HCV IRES translation by more than 97 % for six days. This inhibition was dose dependent. Importantly, a 20 mer with 4 mismatched bases did not show any inhibition. To further evaluate the specificity of the 20 mer oligonucleotide, studies were carried out with reporter plasmids expressing a luciferase gene lacking an IRES or a luciferase gene whose expression was controlled by the encephalomyocarditis virus IRES. Reassuringly, no inhibition of these reporters was observed. In conclusion, we have demonstrated that a 20 mer morpholino oligonucleotide is a potent and specific inhibitor of HCV IRES mediated translation in mice. These studies should aid in the development of therapeutics for the treatment of HCV.
GENE TRANSFER FOR HEMOPHILIA AND OTHER INHERITED DISEASES 478. Development of an AAV-2 Vector for the Treatment of Hemophilia B Thomas C. Harding,1 Kathyrn E. Koprivnikar,1 Melinda J. VanRoey,1 Satya Yendluri,1 Jeff Waugh,1 Brian Donahue.1 1 Research, Cell Genesys, Inc., Foster City, CA, United States. Hemophilia B, the deficiency in the clotting factor IX (FIX), is an Xlinked recessive disorder that occurs in about one in 25,000 males in the U.S. Although the use of plasma-derived or recombinant FIX has greatly extended the life span of these patients, they still remain susceptible to a number of pathologies of the disease including retinopathies, joint bleeding leading to joint dysfunction, vascular occlusive disease, and internal bleeding events that can be life threatening. The use of replication deficient AAV vectors to deliver the human FIX gene offers the opportunity to achieve stable therapeutic levels of FIX in hemophilia patients and the potential to avoid many of these pathologies. We have previously demonstrated the stable expression of human factor IX (hFIX) protein in mice and canine factor IX (cFIX) protein in hemophilic B dogs following gene transfer with a rAAV encoding vector, however, we wished to further optimize the vector transgene cassette used in these studies for tissue specificity and strength of expression. First, several liver-specific promoters were explored and tested. Our rationale was that liver-specific FIX expression would limit gene expression to cells that normally express Factor IX and would help reduce the potential of inducing an anti-hFIX immune response. In addition, the construct was further optimized for expression by the inclusion of additional regulatory elements. Vectors were tested both in cell culture and in vivo for their effect on transgene expression, in comparison to previous generations of AAV-hFIX construct. Results demonstrated that constructs which contained both the b-globin intron and the woodchuck post-transcriptional regulatory element (WPRE) in combination with a liver specific promoter resulted in the strongest expression in vivo, with serum hFIX levels being 5 fold greater than that achieved for an MFG promoter construct. These levels remained stable for over 78 weeks. Although portal vein administration of vector appears to be an effective route of administration to achieve AAV transduction of the liver, it is rather invasive. We therefore also examined alternative delivery routes that maybe more appropriate in the context of hemophilia such as intra-venous (I.V.) and direct liver administration. Results indicate that while the efficiency of gene transfer with I.V. administration of this vector is slightly lower than portal vein administration (70-80% of portal vein values), this route of delivery achieves therapeutic levels of circulating hFIX. Biodistribution was determined by analyses of tissue for the presence of viral vector using real time PCR and demonstrated the predominant organ of transduction was the liver, regardless of administration route. Further studies utilizing this vector will also be presented.
Molecular Therapy Vol. 5, No. 5, May 2002, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy