Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-06-05
2001-05-01
Yucel, Remy (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S002600, C536S023100, C536S023500, C435S006120, C435S007100, C435S091100, C435S069100, C435S366000, C435S455000, C436S513000, C530S300000
Reexamination Certificate
active
06225292
ABSTRACT:
BACKGROUND FOR THE INVENTION
1. Field of the Invention
The invention relates to immunostimulatory sequences in DNA. The invention further relates to recombinant expression vectors for use in gene therapy.
2. History of the Related Art
Recombinant expression vectors are the tools which researchers and clinicians use to achieve the goals of gene therapy and gene immunization. In gene therapy, viral and non-viral vectors are used to deliver an expressible gene into a host to replace a missing or defective gene, or to otherwise supply the host with a therapeutically beneficial polypeptide. In gene immunization, mostly non-viral vectors are used to induce an immune response by the host to an expressed antigen.
One of the obstacles to successful clinical practice of both gene therapy and gene immunization has been the often transient nature of the gene expression achieved in vivo. Transient gene expression is less problematic in gene immunization, where immune responses sufficient for certain immunization schemes may be stimulated by even short-term exposure to expressed antigen. In addition, several options are available to boost the host immune response to antigen, including use of the vector itself as an adjuvant for the desired immune response by exposing the host to non-coding, immunostimulatory nucleotide sequences (ISS-ODN) present in the vector (Sato, et al.,
Science,
273:352-354 (1996)).
However, in a gene therapy protocol, premature loss of gene expression deprives the host of the potential benefits of the therapy (Friedmann,
Scientific American,
“Making Gene Therapy Work” (June 1997)). Repetitive dosing to extend exposure of the host to a therapeutic polypeptide can require that different vectors be used to deliver each dose so the host immune response to vector antigens is minimized (Tripathy, et al.,
Nature Med.,
2:545-550 (1996)).
One potential source of vector immunogenicity are ISS-ODN in the genome of the microbial species used to construct recombinant expression vectors. To explain, the CpG motifs which characterize ISS-ODN are present in bacteria and viruses (including retroviruses) at a much greater frequency than is seen in vertebrate genomes. One consequence of ISS-ODN activity is the ISS-ODN induced production of cytokines such as interferon-&agr; (INF-&agr;), INF-&ggr; and interleukin-12 (IL-12). This ISS-ODN induced inflammation is believed to be defensive against microbial infection in vertebrates and is also believed to be produced in response to ISS-ODN introduced into a host as oligonucleotides or as part of a recombinant expression vector.
SUMMARY OF THE INVENTION
The invention provides compounds consisting of oligodeoxynucleotides, ribonucleotides or analogs thereof which specifically inhibit the immunostimulatory activity of ISS-ODN. ISS-ODN induced secretion of INF-&agr; in particular can suppress recombinant gene expression and directly impedes mRNA and protein synthesis in transfected cells. Thus, inhibition of ISS-ODN activity substantially avoids ISS-ODN induced loss of gene expression, thereby prolonging the availability of the expressed polypeptide to a host undergoing gene therapy or gene immunization with an ISS-ODN containing recombinant expression vector. Further, the need for repetitive dosing to prolong availability of expressed proteins and for extensive reengineering of recombinant expression vectors to eliminate ISS-ODN sequences is avoided through use of the compounds of the invention.
The compounds of the invention are also useful in modulating the immunostimulatory activity of ISS-ODN administered as adjuvants to boost host immune responses to antigen in, for example, immunotherapy. In this respect, the compounds of the invention permit exquisite control over the effect of ISS-ODN based adjuvants in a host.
Further, the compounds of the invention reduce host inflammation generated in response to an infection by an ISS-ODN containing bacteria or virus. Advantageously, the compounds of the invention can be administered to inhibit ISS-ODN activity exerted by a microbe even if the identity of the particular ISS-ODN present in the microbe is unknown. Thus, the compounds of the invention can be considered to be broad spectrum anti-inflammatory agents.
In one aspect, the ISS-ODN inhibitory compounds of the invention are synthesized oligonucleotides (I-ON) which are comprised of the following general primary structures:
5′-Purine—Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3′
or
5′-Purine—Purine-[Y]-[Z]-Pyrmidine-pPyrimidine-3′
where Y is any naturally occurring or synthetic nucleotide except cytosine and is preferably guanosine or inosine (for RNA I-ON). In general, Z is any naturally occurring or synthetic nucleotide or repeat of the same nucleotide. Preferably, when Y is inosine, Z is inosine or one or more guanosine(s). Where Y is guanosine, Z is preferably guanosine or one or more unmethylated cytosine(s). However, when Y is not guanosine or inosine, Z is guanosine or inosine. Most preferably, the 5′ purines are the same nucleotide, as are the 3′ pyrimidines. For example, where ** is YZ, the 5′ purines and 3′ pyrimidines may be AA**TT, AG**TT, GA**TT, GG**TT, AA**TC, AG**TC, and so forth. Any sequences present which flank the hexamer core sequence are constructed to substantially match the flanking sequences present in any known ISS-ODN.
Inhibitory I-ON of the invention are prepared in a pharmaceutically acceptable composition for use in a host. I-ON may be mixed into the composition singly, in multiple copies or in a cocktail of different I-ON. Alternatively, the inhibitory I-ON of the invention may be incorporated into a recombinant expression vector. The inhibitory I-ON can also be provided in the form of a kit, comprising inhibitory I-ON and recombinant expression vector constructs which contain, or are susceptible to insertion of, a gene of interest.
A particular advantage of the I-ON of the invention is that they can be used to target ISS-ODN in any ISS-ODN containing recombinant expression vector or microbe, whether or not the nucleotide composition of the vector or microbe is known. Indeed, it is not necessary that the existence, identity or location of ISS-ODN in the vector or microbe be known. If ISS-ODN are not present in the vector or microbe, the I-ON of the invention will simply have no effect. However, if ISS-ODN are present in the vector or microbe, it can be expected that their immunostimulatory activity will be inhibited in a dose-dependent manner by the I-ON even if the specific structure or location of the ISS-ODN in the vector or microbe is not known.
Thus, in another aspect, the invention provides a simple and effective alternative to the arduous task of eliminating ISS-ODN activity from recombinant expression vectors by identifying all ISS-ODN present in the vector and removing them.
Further in this regard, the invention provides methods for screening recombinant expression vectors for the presence of ISS-ODN and for identifying additional inhibitory I-ON. In the former respect, the presence of ISS-ODN in a recombinant expression vector is confirmed by incubating the vector in a population of lymphocytes with an I-ON of known inhibitory activity and comparing the difference, if any, in the level of ISS-induced cytokine production by the lymphocytes before and after I-ON incubation.
In the latter respect, additional inhibitory I-ON having the characteristics disclosed herein are identified by their ability to inhibit the immunostimulatory activity of a known ISS-containing polynucleotide or recombinant expression vector.
In yet another aspect, the invention further provides a useful anti-inflammatory agent for use in inhibiting the immunostimulatory activity of any ISS-ODN present in an infectious bacterium or virus.
In addition, the invention provides useful means for modulating the immunostimulatory activity of ISS-ODN supplied to a host for immunostimulation (e.g., as an adjuvant).
REFERENCES:
patent: 1234718 (1917-09-01),
Raz Eyal
Roman Mark
Borden Paula A.
Bozicevic Field & Francis LLP
The Regents of the University of California
Yucel Remy
Zara Jane
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