Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-10-23
2001-10-30
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091200, C435S091210
Reexamination Certificate
active
06309819
ABSTRACT:
The present invention relates to a diagnostic system for the detection of Crohn's Disease and ulcerative colitis and to a therapeutic system derived therefrom. No such diagnostic system is currently available.
Until recently there has been no definite cause attributable to the onset of Crohn's Disease or ulcerative colitis which have remained substantially incurable. The best that medicine can provide is some alleviation of the symptoms.
Crohn's Disease afflicts approximately 4 patients per 100,000 of the population, or in the United Kingdom 2,200 new cases per year. Because in its earlier stages Crohn's Disease tends to present as bowel irritation which is much more common, a reliable diagnostic system is required.
Similarly the incidence of ulcerative colitis is sufficient to warrant early diagnosis to enable early treatment.
In Ann. Med. 1993, Volume 25(6), pages 557 to 561, the inventor and a colleage review the evidence for an association between Crohn's Disease and viral infection by various techniques. The inventor and colleagues also discusses observations which suggest measles virus is capable of causing persistent infection of the intestine, and thus Crohn's Disease may be caused by a granulomatous vasculitis in response to the virus in J. Med. Virol. 1993, Volume 39(4), pages 345 to 353.
Virology 1995, Volume 207(1), pages 168-178 addresses measles virus anti-sense sequences in the treatment of cells persistently infected with measles virus.
The applicant has thus shown that the causative agent for Crohn's Disease is the measles virus and accordingly the 32 present invention utilises this finding to provide a diagnostic system for the location of an attenuated vaccine measles virus in bowel tissue, bowel products or in suitable body fluids such as blood or lymph. Continuing studies increasingly support the finding that measles virus is the causative agent for ulcerative colitis. The invention also provides the basis for an anti-viral therapeutic system for the measles virus.
According therefore to a first aspect of the present invention, there is provided a diagnostic system for the detection of Crohn's Disease and ulcerative colitis which system comprises means for detecting attenuated vaccine measles virus RNA or a distinctive metabolic product thereof. Such means may be an antigenic system, or a system utilizing a nucleic acid amplification or hybridization reaction. In the latter case the invention specifically may provide means for performing a reverse transcription polymerase chain reaction or a nucleic acid sequence based amplification reaction or a ligase chain reaction. The systems may including a buffered primer specific for the reverse transcribed DNA from a RNA measles virus and/or an RNA template. The primer may comprise a 5′ modified oligonucleotide sequence specific for any measles virus genomic or antigenomic or messenger RNA. The primer may be attached to a labelling or coagulating moiety such as a fluorochrome for ease of analysis.
The distinctive metabolic product may be selected from a gene sequence or metabolic product thereof to form a nucleocapsid protein, a phosphoprotein, a large protein, an RNA polymerase complex, a matrix protein, a fusion protein or a haemagglutinin protein, specific for the measles virus or a related paramyxovirus.
The nucleotide amplification may be a reverse transcription-polymerase chain reaction (RT-PCR) or nucleic acid sequence based amplification NASBA (or 3SR). In the former case a kit for performing the diagnostic tests as hereinbefore set forth may comprise:
(1) M-MLV reverse transcriptase;
(2) random hexamers and/or oligo (Dt)
12-18
;
(3) a reaction buffer for (1) above;
(4) a PCR reaction buffer including Taq DNA polymerase
(5) a 5′ modified PCR primer with reporter molecules.
Such a kit may further comprise a positive control which comprises measles virus RNA in solution at a known concentration and preferably a negative control or means for its provision.
We have however now found that NASBA is the preferred diagnostic method because it is significantly more sensitive for the detection of the measles virus.
According to a further aspect of the present invention there is provided a method for the in situ analysis of a tissue for Crohn's Disease or ulcerative colitis which method comprises the steps of:
a) obtaining a tissue sample and securing the same in an enclosed reaction container;
b) adding a reagent comprising a PCR buffer; MgCL
2
, dNTP'S, random hexamers and diluting aqueously to a desired dilution factor;
c) subsequently closing the enclosed reaction container and adding M-MLV-RT and subjecting to heat cycling for at least one cycle;
d) treating with a washing buffer;
e) adding a buffered Taq DNA polymerase, MgCL
2
, DNTP and a primer at a predetermined dilution in sterile distilled water and then heat cycling for at least 25 cycles; and
f) subsequently repeating step (d) and viewing for labelled product to indicate the presence of a wild or attenuated vaccine measles virus RNA.
In a further aspect of the present invention, there is provided a method for the in vitro analysis of a sample for Crohn's Disease or ulcerative colitis which method comprises the steps of extracting measles virus RNA from a tissue sample and
a) adding thereto a buffer further comprising DTT, DNTP'S and R Nase inhibitor and oligo (dT)
12-18
and letting down to a desired aqueous concentration in an enclosed reaction container;
b) adding thereto M-MLV-RT and subjecting to incubation to provide a cDNA product;
c) purifying the product and adding thereto a PCR buffer along with MgCL
2
, dNTP's and an outer primer;
d) subsequently adding Taq DNA polymerase and heating for at least 15 cycles;
e) recovering an aliquot of a so-formed reaction production and adding buffered PCR reaction mixture with inner primers and further Taq DNA polymerase and heat cycling for at least 15 cycles; and
f) moving the so-formed product and adding a loading dye and subjecting the resultant product to an electrophoresis to identify a resultant product band;
g) sequencing the amplified products, or hybridising the amplified products with a homologous or hetrologous specific probe to distinguish vaccine strain measles virus from “wild” type measles virus.
According to a further aspect of the present invention there is provided a medicament for the treatment of induced Crohn's Disease and/or ulcerative colitis which method prevents expression, replication, transcription, RNA processing and/or mRNA transport of the measles virus in the host. The medicament may include an attenuated vaccine measles virus antisense RNA for either genomic or antigenomic RNA and a vector. Alternatively, the medicament may comprise an attenuated vaccine measles virus peptide or carbohydrate antigen or a monoclonal or polyclonal antibody thereof. The invention also comprises a measles virus-specific nucleic acid based vaccine genome encoded into an expression system adapted to induce a specific immune response to the attenuated vaccine measles virus and/or its component antigens.
Using antisense RNA to the viral RNA including messenger RNA for the treatment of measles associated diseases alleviates the problem of side effects on host cellular functions. In the past such treatments have proved elusive partially due to the fact that it is extremely difficult to design inhibitors specific for the essential proteins of a virus. However, now in the prior art, the antisense RNA approach has been successfully used to inhibit viral genes of avian retrovirus, Rous sarcoma virus, human immunodificiency virus and simian immunodifeciency virus in cultured cells.
The invention provides a medication which comprises a suitable vector for uptake by the infected cells engineered to carry the appropriate measles virus antisense RNA designed to be targeted against the appropriate gene (e.g Nucleoprotein gene and/or Haemagglutonin gene) followed by a drug resistance gene (e.g Hygromycin B). The method of preparation
Arthur Lisa B.
Licata & Tyrrell P.C.
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