Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1994-05-18
2001-06-12
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S024100
Reexamination Certificate
active
06245560
ABSTRACT:
BACKGROUND OF THE INVENTION
The
Escherichia coli
LTR-7TAR, Bj, rec B-, has been deposited on Jan. 17, 1990 in the American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, Va. 20110 under the accession number 68203.
1. Field of the Invention
The present invention relates to a method of effecting viral inhibition with DNA sequences encoding multiple target response elements, and to constructs suitable for use in same. In particular, the invention relates to a method of inhibiting replication of the Human Immunodeficiency Virus (HIV).
2. Background Information
The tat protein of HIV transactivates viral gene expression and is essential for production of viral products (Arya et al.,
Science
229:69-73 (1985); Sodroski, et al.,
Science
229:74-77 (1985); Dayton, et al.,
Cell
44:941-947 (1986); Fisher, et al.,
Nature
320:367-371 (1986)). The tat activation response element (TAR) has been localized within the region of the first 44 nucleotides downstream of the transcription initiation site (Chen and Okayama,
Mol. Cell. Biol.
7:2745 (1987); Rosen, et al.,
Cell
41:813-823 (1985); Tong-Starksen, et al.,
Proc. Natl. Acad. Sci., USA
84:6845-6849 (1987); Haruber, et al.,
J. Virol.
62:673-679 (1988)). This region, present in all HIV-1 transcripts, forms an unusually stable stem loop structure (Okamoto and Wong-Staal,
Cell
47:29-35 (1986)), and several lines of evidence suggest that the transcriptional effect of tat is mediated through interaction with the TAR region of viral RNA (Sharp, et al.
Cell
59:229-230 (1989); Viscidi, et al.,
Science
246:1606-1608 (1989); Berkhout, et al.,
Cell
59:273-282 (1989); Garcia, et al.,
EMBO J.
8:765-778 (1989); Feng and Holland,
Nature
334:165-167 (1988); Southgate, et al.,
Nature
345:640-642 (1990)).
While tat binding to TAR RNA sequences has been demonstrated (Rappaport, et al., Cold Spring Harbor, N.Y. (1989b); Dingwall, et al.,
Proc. Natl. Acad. Sci. USA
86:6925-6929 (1989)), the sequence requirements for tat binding are not sufficient to explain the sequence and structural requirements needed for transactivation. Cellular factors also appear to play a role in tat mediated transactivation which may confer additional specificity (Marciniak, et al.,
Proc. Natl. Acad. Sci.
87:3624-3628 (1990)). Tat appears to function poorly in nonprimate cells and studies using interspecific hybrids suggest that transactivation potential is correlated with the presence of human chromosome 12 (Hart, et al.,
Science
246:488-491). Several cellular TAR RNA as well as TAR DNA binding proteins have been identified (Gaynor, R. B.
EMBO J.
8:765-778 (1989); Gatignol, et al.,
Proc. Natl. Acad. Sci. USA
86:7828-7832 (1989); Wu, et al.,
EMBO J.
7:2117-2129 (1988); Jones, et al.,
Science
232:755-758 (1986); Garcia, et al.,
EMBO J.
8:765-778 (1989); Marciniak, et al.,
Proc. Natl. Acad. Sci.
87:3624-3628 (1990)), although the role of these proteins in tat mediated transactivation is unclear.
In vitro, tat protein can be released and taken up by cells (Frankel and Pabo,
Cell
55:1189-1193 (1988)), and has biological effects on the regulation of cellular proliferation in addition to its role in HIV promoter activation. Recent studies indicate that tat inhibits antigen-induced lymphocyte proliferation, (Viscidi, et al.,
Science
246:1606-1608 (1989)), has growth promoting activity on cells derived from Kaposi Sarcoma lesions of AIDS patients (Ensoli, et al.,
Nature
340:84-86 (1990)) and stimulates the production of inflammatory cytokines (Buonaguro et al., (1992)
J. Virol.,
66:7159-7167). In contrast, tat does not cause significant reduction of lymphocyte proliferation in response to mitogens. Since the tat protein is critical to HIV replication and the onset of AIDS, interference with tat function will be therapeutically significant.
Transdominant mutations of HIV proteins have been reported (Malim, et al.,
Cell
58:205-214 (1989); Torno, et al.,
Cell
59:113-120 (1989); Marciniak, et al.,
Proc. Natl. Acad. Sci.
87:3624-3628 (1990). These proteins, produced constitutively from a strong promoter, can antagonize the growth of HIV-1 and, therefore, can be used to create cell lines “immunized” to viral infection.
Since TAR RNA appears to interact with tat protein directly (Southgate, et al.,
Nature
345:640-624 (1990)) or through the combined activities of cellular factor(s) (Marciniak, et al.,
Proc. Natl. Acad. Sci. USA
87:3624-3628 (1990), we hypothesized that TAR RNA, produced in large amounts, might serve as a competitive inhibitor of tat function.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a DNA construct having a vector and a promoter operably linked to at least two DNA segments encoding HIV-1 tat activation response (TAR) elements so that the elements are transcribed in tandem. Preferably, the promoter is a primate lentivirus LTR. Most preferably, the LTR is the Human Immunodeficiency Virus-1 (HIV-1) LTR. Advantageously, the DNA construct between 5 and 50 TAR elements. In addition, the DNA construct the promoter in this embodiment can preferably be regulated by the HIV-1 tat protein. A preferred vector in this embodiment is pCD7. More preferably, the vector is a retroviral vector and the retroviral vector is either DC-antitat/30 or DC-antitat/23. Still more preferably, the vector has a DNA segment encoding tat antisense RNA. Most preferably, the tat antisense RNA and the TAR sequences are on the same RNA molecule.
Another embodiment of the present invention is a method of treating lentivirus infection. This method has the following steps.
(i) obtaining cells from a lentivirus-infected patient;
(ii) introducing the lentivirus-inhibiting construct according to Claim
1
into the cells;
(iii) expanding the cells in culture; and
(iv) reintroducing cells resulting from step (iii) to the patient under conditions such that the treatment is effected.
Advantageously, the lentivirus in this method is HIV-1. In addition, the cells can be bone marrow cells or blood cells. More preferably, the blood cells are CD4
+
T-cells. In an even more advantageous embodiment, the number of the CD4
+
T-cells increases as a result of the introduction of the construct. In this method, the expanding step might involve treatment with a mitogen, with a preferable mitogen being phytohemagglutinin. During this method, the cells can be cultured in the presence of a cytokine, such as interleukin-2, after treatment with the mitogen. Also, during this method, the construct introduced in step (ii) can be a DNA construct having a vector with a promoter operably linked to at least two HIV-1 TAR elements and to a sequence encoding a molecule that inhibits lentivirus replication, wherein the TAR elements and the sequence are transcribed together in the same transcript.
Yet another embodiment of the present invention is a method for protecting against lentivirus infection by the steps of:
(i) obtaining cells from an asymptomatic individual;
(ii) introducing the lentivirus-inhibiting construct according to Claim
1
into the cells;
(iii) expanding the cells; and
(iv) reintroducing cells resulting from step (iii) to the individual under conditions such that the treatment is effected.
Advantageously, the lentivirus in this method can be HIV-1.
Still another embodiment of the present invention is a method of inhibiting lentivirus replication by introducing into lentivirus-infected cells the DNA construct discussed above under conditions such that inhibition is effected. Most preferably, the lentivirus is HIV.
Yet a further embodiment of the present invention is a method of inhibiting lentivirus replication by introducing into a lentivirus-infected cell the DNA construct discussed above wherein a lentivirus protein product regulates transcription of the lentivirus LTR so that the inhibition is effected. The lentivirus in this case is advantageously HIV.
A further embodiment of the present invention is a DNA construct having a vector with a promoter operably linked to at least two HIV-1 TAR elements and to a sequence
Knobbe Martens Olson & Bear LLP
The United States of America as represented by the Department of
Yucel Remy
LandOfFree
Vector with multiple target response elements affecting gene... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Vector with multiple target response elements affecting gene..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Vector with multiple target response elements affecting gene... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2501903