Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-03-19
2001-06-12
Martin, Jill D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S008000, C800S021000, C800S022000, C800S025000, C435S455000, C435S456000, C435S320100, C435S325000
Reexamination Certificate
active
06245966
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to a method of introducing exogenous genetic materials into lymphocytes, in vitro and in vivo, and its use in studying the function and regulation of immune system. Specifically, lymphocytes are genetically engineered to express a truncated Coxsackievirus and/or Adenovirus Receptor (tCAR) protein to allow improved adenovirus vector transfection.
The inefficiency of gene delivery into lymphocytes has hampered the investigation of the pathways that control lymphocyte physiology. Although retrovirus vectors have been used successfully to transduce multiple hematopoietic cell types, experiments involving retroviral transduction of lymphocytes are limited by the difficulty in transducing the entire population of cells, the requirement that the cells be proliferating for viral integration, and the time required for the transduction and expression of the introduced gene. Other approaches to manipulate gene expression in lymphocyte cell lines often require either the generation of cell lines that express the desired gene product, sometimes under the control of an inducible promoter, or the transient transfection of the gene of interest into a fraction of the cells. In addition, the manipulation of gene expression in vivo usually entails the creation of transgenic or gene disrupted mice. These technologies, however, are costly and time consuming, thus have been applied to a few limited cases. Furthermore, the expression of an exogenous gene in transgenic mice is often detrimental to the normal development. The inability to introduce genes easily and efficiently into lymphocytes and examine the consequences of such expression soon after gene delivery limits the characterization of lymphocyte pathways that control cell growth, differentiation and death.
Adenoviral vectors are attractive in that either proliferating or quiescent cells can be transduced, the expression of the introduced gene is evident by as early as 5 hrs after transfection, the vectors can accommodate large insert sizes (7-9 kilobases), and high titer stocks are easily generated [Nevins, J. R. et al., (1997)
Meth. Enzymol
283:205-219]. A particular advantage of the adenovirus vectors lies in their ability to transduce the entire cell population. Group C adenovirus (e.g. Ad2 and Ad5) infection requires the high affinity attachment of the viral fiber capsid protein to a cellular receptor and viral penton base binding to certain cellular integrins, followed by cell entry via receptor-mediated endocytosis [Wickham, T. J., et al., (1993)
Cell
73:309-319]. Unfortunately, although adenovirus can infect a wide range of cell types, lymphocytes are not very susceptible to adenovirus infection, apparently as a result of the failure of adenovirus to efficiently bind the cell surface and be internalized [DeMatteo, R. P. et al., (1997)
Ann. of Surgery
222:229-242; Neering, S. J. et al., (1996)
Blood
88:1147-1155; Chu, Y. et al., (1992)
Virology
188:793-800]. In part, the inability of adenovirus to enter T cells is due to the very low levels of cellular fiber receptor expressed on these cells [Huang, S. et al., (1997)
Journal of Virology
69:2257-2263; Huang, S. et al., (1996)
J. Virol.
70:4502-4508]. In addition, T cells express limiting levels of &agr;V containing integrins, and mitogen-mediated upregulation of &agr;V integrin expression confers limited infection by adenovirus vectors [Huang et al., (1997) supra].
The cDNA for the cellular receptor for the adenoviral fiber protein, CAR (for Coxsackievirus and/or Adenovirus Receptor), was recently cloned, and the expression of CAR in Chinese hamster ovary (CHO) cells increased their susceptibility to adenovirus infection approximately 100 fold [Bergelson, J. M. et al., (1997)
Science
275:1320-1323].
There has been a long-felt need in the art for an efficient gene transfer method for lymphocytes. The invention described herein provides in vitro cell culture systems and in vivo animal models allowing efficient adenovirus-mediated transduction, and subsequently, enabling the art to study diverse functions of lymphocytes as well as to test agents to modulate lymphocyte activity.
SUMMARY OF THE INVENTION
In the context of the present invention, a truncated form of CAR (tCAR), lacking substantially all of the cytoplasmic domain, has adenovirus and/or Coxsackie virus binding activity but yet no adverse effect on integrin expression when it (tCAR) is expressed in lymphocytes in culture or in vivo. With reference to SEQ ID NO:1, mature human tCAR has an amino acid sequence extending from 1 to between about 262 and 285; as specifically exemplified, mature tCAR has 262 amino acids.
The present invention further provises a method for generating lymphocyte cell lines which are susceptible for adenoviral transduction. Specifically, lymphocytes in vitro are genetically engineered using retroviral expression vectors to express the truncated form of CAR (tCAR) stably and thus become high efficiency target cells for adenoviral transduction.
The present invention also provides a transgenic mouse which expresses truncated CAR in its lymphocytes and is thus susceptible for adenoviral transduction in a tissue specific manner. These animals can be used for targeted gene transfer into lymphocytes using adenoviral vectors in vivo or ex vivo.
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patent: 98/11221 (1998-03-01), None
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Chu, Y. et al. “Persistent Infection of Human Adenovirus Type 5 in Human Monocyte Cell Lines”; (1992)Virology188:793-800.
Huang, S. et al. “Upregulation of Integrins &agr;v&bgr;3 and &agr;v&bgr;5 on Human Monocytes and T Lymphocytes Facilitates Adenovirus-Mediated Gene Delivery”; (1997)Journal of Virology69:2257-2263.
Leon, R.P. et al. “Adenoviral-mediated gene transfer in lymphocytes”; (1998)Proc. Natl. Acad. Sci. USA95:13159-13164.
Miller, A.D. et al. “Improved Retroviral Vectors for Gene Transfer and Expression” (1989)Biotechniques7:980-988.
Nevins, J.R. et al. “Functional Analysis of E2F Transcription Factor”(1997)Meth. Enzymol283:205-219.
Smith et al. “CrmA expression in T lymphocytes of transgenic mice inhibits CD95 (Fas/APO-1)-transduced apoptosis, but does not cause lymphadenopathy or autoimmune disease”; (1996) EMBO J. 15:5167-5176.
Wang, X. et al. “Coxsackievirus and Adenovirus Receptor Cytoplasmic and Transmembrane Domains Are Not Essential for Coxsackievirus and Adenorivus Infection”; (1999)Journal of Virology73(3):2559-2562.
Wickham, T,J. et al. “Integrins &agr;v&bgr;3and &agr;v&bgr;5Promote Adenovirus Internalization but Not Virus Attachment”; (1993)Cell73:309-319.
Greenlee Winner and Sullivan P.C.
Martin Jill D.
University Technology Corporation
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