Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

Background: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the trans...

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Veröffentlicht in:Journal of thrombosis and haemostasis 2011-11, Vol.9 (11), p.2235-2242
Hauptverfasser: PIPE, S. W., MIAO, H., BUTLER, S. P., CALCATERRA, J., VELANDER, W. H.
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container_end_page 2242
container_issue 11
container_start_page 2235
container_title Journal of thrombosis and haemostasis
container_volume 9
creator PIPE, S. W.
MIAO, H.
BUTLER, S. P.
CALCATERRA, J.
VELANDER, W. H.
description Background: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods: Transgenic mice were made with a mammary‐specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS‐PAGE, western blot, and one‐stage and two‐stage clotting assays. The hemostatic activity of immunoaffinity‐enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single‐chain molecule that retained high specific activity, similar to therapeutic‐grade FVIII. 226/N6 had > 450‐fold higher IU mL−1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma‐derived VWF as therapeutic‐grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof‐of‐principle for the study of expression of 226/N6 and perhaps other single‐chain bioengineered rFVIIIs in the milk of transgenic livestock.
doi_str_mv 10.1111/j.1538-7836.2011.04505.x
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W. ; MIAO, H. ; BUTLER, S. P. ; CALCATERRA, J. ; VELANDER, W. H.</creator><creatorcontrib>PIPE, S. W. ; MIAO, H. ; BUTLER, S. P. ; CALCATERRA, J. ; VELANDER, W. H.</creatorcontrib><description>Background: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods: Transgenic mice were made with a mammary‐specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS‐PAGE, western blot, and one‐stage and two‐stage clotting assays. The hemostatic activity of immunoaffinity‐enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single‐chain molecule that retained high specific activity, similar to therapeutic‐grade FVIII. 226/N6 had &gt; 450‐fold higher IU mL−1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma‐derived VWF as therapeutic‐grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof‐of‐principle for the study of expression of 226/N6 and perhaps other single‐chain bioengineered rFVIIIs in the milk of transgenic livestock.</description><identifier>ISSN: 1538-7933</identifier><identifier>ISSN: 1538-7836</identifier><identifier>EISSN: 1538-7836</identifier><identifier>DOI: 10.1111/j.1538-7836.2011.04505.x</identifier><identifier>PMID: 21920013</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Animals ; Bioengineering ; Cells, Cultured ; Factor VIII - biosynthesis ; Factor VIII - genetics ; Factor VIII - therapeutic use ; factor VIII ; hemophilia ; Hemophilia A - drug therapy ; Humans ; mammary gland ; Mice ; Mice, Transgenic ; Milk - chemistry ; Milk - metabolism ; modified B domain ; recombinant protein ; transgenic animal ; von Willebrand factor ; von Willebrand Factor - biosynthesis ; von Willebrand Factor - genetics</subject><ispartof>Journal of thrombosis and haemostasis, 2011-11, Vol.9 (11), p.2235-2242</ispartof><rights>2011 International Society on Thrombosis and Haemostasis</rights><rights>2011 International Society on Thrombosis and Haemostasis.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5065-a671fd0857cff80824844a5620cc3ce4d48ee0b76df6bf4cdb37c86a753db7d93</citedby><cites>FETCH-LOGICAL-c5065-a671fd0857cff80824844a5620cc3ce4d48ee0b76df6bf4cdb37c86a753db7d93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21920013$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>PIPE, S. W.</creatorcontrib><creatorcontrib>MIAO, H.</creatorcontrib><creatorcontrib>BUTLER, S. P.</creatorcontrib><creatorcontrib>CALCATERRA, J.</creatorcontrib><creatorcontrib>VELANDER, W. H.</creatorcontrib><title>Functional factor VIII made with von Willebrand factor at high levels in transgenic milk</title><title>Journal of thrombosis and haemostasis</title><addtitle>J Thromb Haemost</addtitle><description>Background: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods: Transgenic mice were made with a mammary‐specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS‐PAGE, western blot, and one‐stage and two‐stage clotting assays. The hemostatic activity of immunoaffinity‐enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single‐chain molecule that retained high specific activity, similar to therapeutic‐grade FVIII. 226/N6 had &gt; 450‐fold higher IU mL−1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma‐derived VWF as therapeutic‐grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof‐of‐principle for the study of expression of 226/N6 and perhaps other single‐chain bioengineered rFVIIIs in the milk of transgenic livestock.</description><subject>Animals</subject><subject>Bioengineering</subject><subject>Cells, Cultured</subject><subject>Factor VIII - biosynthesis</subject><subject>Factor VIII - genetics</subject><subject>Factor VIII - therapeutic use</subject><subject>factor VIII</subject><subject>hemophilia</subject><subject>Hemophilia A - drug therapy</subject><subject>Humans</subject><subject>mammary gland</subject><subject>Mice</subject><subject>Mice, Transgenic</subject><subject>Milk - chemistry</subject><subject>Milk - metabolism</subject><subject>modified B domain</subject><subject>recombinant protein</subject><subject>transgenic animal</subject><subject>von Willebrand factor</subject><subject>von Willebrand Factor - biosynthesis</subject><subject>von Willebrand Factor - genetics</subject><issn>1538-7933</issn><issn>1538-7836</issn><issn>1538-7836</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1O3DAUhS1UxF95BeRdu5lwHduJs2ilCpUyCIkNbdWV5djOjKeOQ-PMAG_Ds_BkOMwwopuq3vha57vnXusghAlkJJ3TRUY4FZNS0CLLgZAMGAee3e-gg63w7rWuKN1HhzEuAEjFc9hD-zmp8vSiB-jX-TLowXVBedwoPXT90-OP6XSKW2UsvnPDHK-6gH86723dq2A2FFYDnrvZHHu7sj5iF_CQ5DizwWncOv_7PdptlI_2eHMfoe_nX2_OLiZX19-mZ1-uJppDwSeqKEljQPBSN40AkTPBmOJFDlpTbZlhwlqoy8I0Rd0wbWpaalGoklNTl6aiR-jz2vd2WbfWaBvSIl7e9q5V_YPslJN_K8HN5axbScoYS9OSwYeNQd_9Wdo4yNZFbb1XwXbLKCsgBaMlsER-_CdJaFVVQEGQhIo1qvsuxt4224UIyDFDuZBjPHKMSo4ZypcM5X1qPXn7oW3ja2gJ-LQG7py3D_9tLC9vLsaKPgOUJayf</recordid><startdate>201111</startdate><enddate>201111</enddate><creator>PIPE, S. W.</creator><creator>MIAO, H.</creator><creator>BUTLER, S. P.</creator><creator>CALCATERRA, J.</creator><creator>VELANDER, W. H.</creator><general>Blackwell Publishing Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>201111</creationdate><title>Functional factor VIII made with von Willebrand factor at high levels in transgenic milk</title><author>PIPE, S. W. ; MIAO, H. ; BUTLER, S. P. ; CALCATERRA, J. ; VELANDER, W. H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5065-a671fd0857cff80824844a5620cc3ce4d48ee0b76df6bf4cdb37c86a753db7d93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Animals</topic><topic>Bioengineering</topic><topic>Cells, Cultured</topic><topic>Factor VIII - biosynthesis</topic><topic>Factor VIII - genetics</topic><topic>Factor VIII - therapeutic use</topic><topic>factor VIII</topic><topic>hemophilia</topic><topic>Hemophilia A - drug therapy</topic><topic>Humans</topic><topic>mammary gland</topic><topic>Mice</topic><topic>Mice, Transgenic</topic><topic>Milk - chemistry</topic><topic>Milk - metabolism</topic><topic>modified B domain</topic><topic>recombinant protein</topic><topic>transgenic animal</topic><topic>von Willebrand factor</topic><topic>von Willebrand Factor - biosynthesis</topic><topic>von Willebrand Factor - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>PIPE, S. W.</creatorcontrib><creatorcontrib>MIAO, H.</creatorcontrib><creatorcontrib>BUTLER, S. P.</creatorcontrib><creatorcontrib>CALCATERRA, J.</creatorcontrib><creatorcontrib>VELANDER, W. H.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of thrombosis and haemostasis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>PIPE, S. W.</au><au>MIAO, H.</au><au>BUTLER, S. P.</au><au>CALCATERRA, J.</au><au>VELANDER, W. H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Functional factor VIII made with von Willebrand factor at high levels in transgenic milk</atitle><jtitle>Journal of thrombosis and haemostasis</jtitle><addtitle>J Thromb Haemost</addtitle><date>2011-11</date><risdate>2011</risdate><volume>9</volume><issue>11</issue><spage>2235</spage><epage>2242</epage><pages>2235-2242</pages><issn>1538-7933</issn><issn>1538-7836</issn><eissn>1538-7836</eissn><abstract>Background: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods: Transgenic mice were made with a mammary‐specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS‐PAGE, western blot, and one‐stage and two‐stage clotting assays. The hemostatic activity of immunoaffinity‐enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single‐chain molecule that retained high specific activity, similar to therapeutic‐grade FVIII. 226/N6 had &gt; 450‐fold higher IU mL−1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma‐derived VWF as therapeutic‐grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof‐of‐principle for the study of expression of 226/N6 and perhaps other single‐chain bioengineered rFVIIIs in the milk of transgenic livestock.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>21920013</pmid><doi>10.1111/j.1538-7836.2011.04505.x</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record>
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source MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection
subjects Animals
Bioengineering
Cells, Cultured
Factor VIII - biosynthesis
Factor VIII - genetics
Factor VIII - therapeutic use
factor VIII
hemophilia
Hemophilia A - drug therapy
Humans
mammary gland
Mice
Mice, Transgenic
Milk - chemistry
Milk - metabolism
modified B domain
recombinant protein
transgenic animal
von Willebrand factor
von Willebrand Factor - biosynthesis
von Willebrand Factor - genetics
title Functional factor VIII made with von Willebrand factor at high levels in transgenic milk
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