Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-04
2003-11-04
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S320100, C435S212000, C514S04400A
Reexamination Certificate
active
06642028
ABSTRACT:
BACKGROUND OF THE INVENTION
Recombinant DNA technology is currently the most valuable tool known for producing highly pure therapeutic proteins both in vitro and in vivo to treat clinical diseases. Accordingly, a vast number of genes encoding therapeutic proteins have been identified and cloned to date, providing valuable sources of protein. The value of these genes is, however, often limited by low expression levels.
This problem has traditionally been addressed using regulatory elements, such as optimal promoters and enhancers, which increase transcription/expression levels of genes. Additional techniques, particularly those which do not rely on foreign sequences (e.g., viral or other foreign regulatory elements) for increasing transcription efficiency of cloned genes, resulting in higher expression, would be of great value.
Accordingly, the present invention provides novel methods for increasing gene expression, and novel genes which exhibit such increased expression.
Gene expression begins with the process of transcription. Factors present in the cell nucleus bind to and transcribe DNA into RNA. This RNA (known as pre-mRNA) is then processed via splicing to remove non-coding regions, referred to as introns, prior to being exported out of the cell nucleus into the cytoplasm (where they are translated into protein). Thus, once spliced, pre-mRNA becomes mRNA which is free of introns and contains only coding sequences (i.e., exons) within its translated region.
Splicing of vertebrate pre-mRNAs occurs via a two step process involving splice site selection and subsequent excision of introns. Splice site selection is governed by definition of exons (Berget et al. (1995)
J. Biol. Chem
. 270(6):2411-2414), and begins with recognition by splicing factors, such as small nuclear ribonucleoproteins (snRNPs), of consensus sequences located at the 3′ end of an intron (Green et al. (1986)
Annu. Rev. Genet
. 20:671-708). These sequences include a 3′ splice acceptor site, and associated branch and pyrimidine sequences located closely upstream of 3′ splice acceptor site (Langford et al. (1983)
Cell
33:519-527). Once bound to the 3′ splice acceptor site, splicing factors search downstream through the neighboring exon for a 5′ splice donor site. For internal introns, if a 5′ splice donor site is found within about 50 to 300 nucleotides downstream of the 3′ splice acceptor site, then the 5′ splice donor site will generally be selected to define the exon (Robberson et al. (1990)
Mol. Cell. Biol
. 10(1):84-94), beginning the process of spliceosome assembly.
Accordingly, splicing factors which bind to 3′ splice acceptor and 5′ splice donor sites communicate across exons to define these exons as the original units of spliceosome assembly, preceding excision of introns. Typically, stable exon complexes will only form and internal introns thereafter be defined if the exon is flanked by both a 3′ splice acceptor site and 5′ splice donor site, positioned in the correct orientation and within 50 to 300 nucleotides of one another.
It has also been shown that the searching mechanism defining exons is not a strict 5′ to 3′ (i.e., downstream) scan, but instead operates to find the “best fit” to consensus sequence (Robberson et al., supra. at page 92). For example, if a near-consensus 5′ splice donor site is located between about 50 to 300 nucleotides downstream of a 3′ splice acceptor site, it may still be selected to define an exon, even if it is not consensus. This may explain the variety of different splicing patterns (referred to as “alternative splicing”) which is observed for many genes.
SUMMARY OF THE INVENTION
The present invention provides novel DNAs which exhibit increased expression of a protein of interest. The novel DNAs also can be characterized by increased levels of cytoplasmic mRNA accumulation following transcription within a cell, and by novel splicing patterns. The present invention also provides expression vectors which provide high tissue-specific expression of DNAs, and compositions for delivering such vectors to cells. The invention further provides methods of increasing gene expression and/or modifying the transcription pattern of a gene. The invention still further provides methods of producing a protein by recombinant expression of a novel DNA of the invention.
In one embodiment, a novel DNA of the invention comprises an isolated DNA (e.g., gene clone or cDNA) containing one or more consensus or near consensus splice sites (3′ splice acceptor or 5′ splice donor) which have been corrected. Such consensus or near consensus splice sites can be corrected by, for example, mutation (e.g., substitution) of at least one consensus nucleotide with a different, preferably non-consensus, nucleotide. These consensus nucleotides can be located within a consensus or near consensus splice site, or within an associated branch sequence (e.g., located upstream of a 3′ splice acceptor site). Preferred consensus nucleotides for correction include invariant (i.e., conserved) nucleotides, including one or both of the invariant bases (
AG
) present in a 3′ splice acceptor site; one or both of the invariant bases (
GT
) present in a 5′ splice donor site; or the invariant
A
present in the branch sequence of a 3′ splice acceptor site.
If the consensus or near consensus splice site is located within the coding region of a gene, then the correction is preferably achieved by conservative mutation. In a particularly preferred embodiment, all possible conservative mutations are made within a given consensus or near consensus splice site, so that the consensus or near consensus splice site is as far from consensus as possible (i.e., has the least homology to consensus as is possible) without changing the coding sequence of the consensus or near consensus splice site.
In another embodiment, a novel DNA of the invention comprises at least one non-naturally occurring intron, either within a coding sequence or within a 5′ and/or 3′ non-coding sequence of the DNA. Novel DNAs comprising one or more non-naturally occurring introns may further comprise one or more consensus or near consensus splice sites which have been corrected as previously summarized.
In a particular embodiment of the invention, the present invention provides a novel gene encoding a human Factor VIII protein. This novel gene comprises one or more non-naturally occurring introns which serve to increase transcription of the gene, or to alter splicing of the gene. The gene may alternatively or additionally comprise one or more consensus splice sites or near consensus splice sites which have been corrected, also to increase transcription of the gene, or to alter splicing of the gene. In one embodiment, the Factor VIII gene comprises the coding region of the full-length human Factor VIII gene, except that the coding region has been modified to contain an intron spanning, overlapping or within the region of the gene encoding the &bgr;-domain. This novel gene is therefore expressed as a &bgr;-domain deleted human Factor VIII protein, since all or a portion of the &bgr;-domain coding sequence (defined by an intron) is spliced out during transcription.
A particular novel human Factor VIII gene of the invention comprises the nucleotide sequence shown in SEQ ID NO:1. Another particular novel human Factor VIII gene of the invention comprises the coding region of the nucleotide sequence shown in SEQ ID NO:3 (nucleotides 1006-8237). Particular novel expression vectors of the invention comprise the complete nucleotide sequences shown in SEQ ID NOS: 2, 3 and 4. These vectors include novel 5′ untranslated regulatory regions designed to provide high liver-specific expression of human Factor VIII protein.
In still other embodiments, the invention provides a method of increasing expression of a DNA sequence (e.g., a gene, such as a human Factor VIII gene), and a method of increasing the amount of mRNA which accumul
Bidlingmaier Scott
Gonzales Jose E. N.
Ill Charles R.
Yang Claire Q.
Lahive & Cockfield LLP
Low Christopher S. F.
Remillard, Esq. Jane E.
Schnizer Holly
The Immune Response Corporation
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