Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-05-01
2003-07-29
Falk, Anne-Marie (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S325000, C435S340000, C435S070100, C536S024100
Reexamination Certificate
active
06599744
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to improved vectors, and more specifically, improved adenovirus vectors useful for gene therapy.
BACKGROUND
Adenoviruses (Ad) are double-stranded DNA viruses. The genome of adenoviruses (~36 kb) is complex and contains over 50 open reading frames (ORFs). These ORFs are overlapping and genes encoding one protein are often embedded within genes coding for other Ad proteins. Expression of Ad genes is divided into an early and a late phase. Early genes are those transcribed prior to replication of the genome while late genes are transcribed after replication. The early genes comprise E1a, E1b, E2a, E2b, E3 and E4. The E1a gene products are involved in transcriptional regulation; the E1b gene products are involved in the shut-off of host cell functions and MRNA transport. E2a encodes the a DNA-binding protein (DBP); E2b encodes the viral DNA polymerase and preterminal protein (pTP). The E3 gene products are not essential for viral growth in cell culture. The E4 region encodes regulatory protein involved in transcriptional and post-transcriptional regulation of viral gene expression; a subset of the E4 proteins are essential for viral growth. The products of the late genes (e.g., L1-5) are predominantly components of the virion as well as proteins involved in the assembly of virions. The VA genes produce VA RNAs which block the host cell from shutting down viral protein synthesis.
Adenoviruses or Ad vectors have been exploited for the delivery of foreign genes to cells for a number of reasons including the fact that Ad vectors have been shown to be highly effective for the transfer of genes into a wide variety of tissues in vivo and the fact that Ad infects both dividing and non-dividing cells; a number of tissues which are targets for gene therapy comprise largely non-dividing cells.
The current generation of Ad vectors suffer from a number of limitations which preclude their widespread clinical use. The most serious limitation is the loss of expression of genes of interest in cells infected with Ad vectors. It has been assumed that this loss in expression is due to immune detection and elimination of cells infected with Ad vectors, but more recently transcriptional regulation has been raised as a potential factor in loss of transgene expression.
What is needed is an approach that overcomes the problem of loss of expression of genes of interest in cells infected with Ad vectors. Such an approach should ensure long-term expression for gene therapy and other applications.
SUMMARY OF THE INVENTION
The present invention contemplates improving vectors generally, and more specifically improving adenovirus vectors. The present invention contemplates both improved compositions (e.g., expression vectors) and methods (e.g., methods of transfection and gene therapy). With regard to compositions, the present invention contemplates an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest. The invention is not limited by the nature of the ribosomal promoter sequence chosen; any non-viral promoter sequence or portion thereof which is functional in cells (i.e., such that a gene of interest can be expressed and/or overexpressed) may be utilized. A variety of ribosomal promoters are known to those skilled in the art. Preferred ribosomal promoters are eukaryotic ribosomal promoters, including but not limited to mammalian ribosomal promoters such as those of mice, rats, rabbits, pigs and humans.
In a preferred embodiment, said expression vector further comprises viral nucleic acid. It is not intended that the expression vector be limited to a particular viral vector. In one embodiment, said expression vector further comprises adenoviral nucleic acid. Indeed, the present invention contemplates replication-defective adenoviral vectors comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products. In a preferred embodiment, adenoviral vectors shown to be free of E1 function (e.g., by absence of replication on HeLA cells) are contemplated, such vectors comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products.
The present invention also contemplates a mammalian cell line containing the above-described recombinant vector and integrated viral sequences expressing E1 function. It is preferred that said cell line is a 293-derived cell line.
With regard to methods of transfection, the present invention contemplates a method, comprising: a) providing: i) eukaryotic cells, ii) an expression vector comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products; and b) introducing said expression vector into said cells. Again, in a preferred embodiment, said expression vector further comprises viral nucleic acid, such as adenoviral nucleic acid.
With regard to methods of gene therapy, in one embodiment, the present invention contemplates a method for delivering nucleic acid to cells of an animal, comprising: a) providing: i) an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest, ii) a recipient animal; and b) administering said vector to said recipient animal.
In a particular embodiment, the oligonucleotide or gene cassette is delivered to a particular tissue in said animal. It is not intended that the present invention be limited to the particular tissue type. In one embodiment, however, said tissue is selected from the group consisting of lung, trachea and liver tissue. For delivery to the liver, the hepatocytes can be readily transfected in vivo by direct vector infusion in the portal vein, as well as to the peripheral circulation.
With regard to other methods of gene therapy, in another embodiment, the present invention contemplates a method for delivering nucleic acid to cells of an animal, comprising: a) providing: i) an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest, ii) a recipient animal; b) coupling said expression vector to a carrier to generate a composition; and c) administering said composition to said recipient animal.
Where adenoviral vectors are employed in the present invention it is not intended that the present invention be limited by the precise size of the vector, although it is generally desirable that the vector have a total size of between 20 and 40 kilobase pairs. It is preferred that the total size of the DNA packaged into an adenovirus particle derived from these vectors is about the length of the wild-type adenovirus genome (~36 kb). It is well known in the art that DNA representing about 105% of the wild-type length may be packaged into a viral particle; thus the adenovirus particle derived from recombinant vector may contain DNA whose length exceeds by ~105% the size of the wild-type genome. The size of the recombinant plasmid may be adjusted using reporter genes and genes of interest having various sizes (including the use of different sizes of introns within these genes) as well as through the use of irrelevant or non-coding DNA fragment which act as “stuffer” fragments (e.g., portions of bacteriophage genomes).
In one embodiment, the present invention contemplates recovering said encapsidated adenovirus minichromosome and, in turn, purifying said recovered encapsidated adenovirus minichromosome. Thereafter, said purified encapsidated adenovirus minichromosome can be administered to a host (e.g. a mammal). Human therapy is thereby contemplated.
It is not intended that the present invention be limited by the nature of the administration of said adenovirus minichromosomes. All types of administration are contemplated, including direct injection (intramuscular, intravenous, subcutaneous, etc.), inhalation, etc.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1
shows mouse L32 ribosomal promoter sequences (SEQ ID NO:1).
FIG. 2
shows pig ribosomal promoter sequences (SEQ ID NO:2).
FIG. 3
shows the results of in vivo transfection of liver of recipient anima
Falk Anne-Marie
Medlen & Carroll LLP
Regents of the University of Michigan
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