Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-12-16
2001-05-15
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S325000, C435S455000, C536S023100, C536S024100
Reexamination Certificate
active
06232105
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the field of molecular biology, in particular the field of systems for the replication, transcription and/or expression (especially translation into protein) of genes or other nucleic acid molecules of interest.
BACKGROUND OF THE INVENTION
So many of these systems have been developed over the last two or three decades that it is hardly feasible to give a useful summary of the many possibilities. These possibilities are generally known to the people skilled in this art anyway. However, there are a number of genes which are difficult to replicate, transcribe or express for a variety of reasons. A quite obvious reason is for instance that the product produced upon expression is toxic to the cell in which the nucleic acid of interest is expressed. There are however less clear reasons why replication, transcription or expression of a nucleic acid of interest does not lead to useful levels of replication, transcription and/or expression. This invention typically deals with the replication, transcription and/or expression of such nucleic acids. The present invention was made during research involving adeno associated virus (AAV) and is typically useful for replication, transcription and/or expression of nucleic acids in an AAV-based system and typically for replication, transcription and/or expression of AAV-genes, in particular the cap-gene. However, other genes resisting replication, transcription and/or expression in the regular systems or genes or other nucleic acids that may only be produced upon induction will also be suitable for use in the presently invented system. The invention will however ne explained in more detail based on the AAV-system. AAV is a virus that is typically suggested for use in gene therapy, whereby a gene of interest is packaged into an AAV virion, which can infect a cell to be provided with said gene. The present invention arrives at a universal packaging system for AAV derived vectors provided with such a gene therapy related nucleic acid.
AAV is a non-pathogenic human parvovirus (reviewed in
1, 2
). The virus replicates as a single strand DNA of approximately 4.6 kb. Both the plus and the minus strand are packaged and infectious. Efficient replication of AAV requires the co-infection of the cell by a helper virus such as Adenovirus or Herpes Simplex Virus. In the absence of a helper virus, no substantial replication of AAV is observed. AAV is therefore also classified as a “Dependovirus”, When no helper virus is present, the AAV genome can integrate into the host cell genome. The wild-type virus has a strong preference (70%) for an integration site on the long arm of chromosome 19 (19 q13.3)
3-5
. Following integration, the expression of the virus genes is not detectable. The integrated provirus replicates as a normal part of the host cell genome upon division of the transduced cell and ends up in both daughter cells. This stage of the virus life cycle is known as the latent stage. This latent stage is stable but can be interrupted by infection of the transduced cell by a helper virus. Following infection of the helpervirus, AAV is excised from the host cell genome and starts to replicate. During the early phase of this lytic cycle the rep-genes are expressed. Approximately 12 to 16 hours later, the capsid proteins VP1, VP2 and VP3 are produced and the replicated virus DNA is packaged into virions (structure of the AAV-genome and its genes is depicted in FIG.
1
). The virions accumulate in the nucleus of the cell and are released when the cell lyses as a result of the accumulation of AAV and the helpervirus (reviewed in
1, 2
).
The AAV-genome contains two genes named rep and cap (FIG.
1
). Three promoters (P5, P19 and P40) drive the synthesis of mRNAs coding for 4 Rep-proteins (Rep78, Rep68, Rep52 and Rep40) and three capsid proteins (VP1, VP2 and VP3). The AAV-genome is flanked on both sides by a 145 bp sequence, called the Inverted Terminal Repeat (ITR), which appears to contain all the cis-acting sequences required for virus integration, replication and encapsidation
6, 7
.
The capsid proteins VP1, VP2 and VP3 are produced from a 2.6 kb transcript of the AAV P40 promoter, which is spliced into two 2.3 kb mRNAs by using the same splice donor but two different splice acceptor sites. The splice acceptor sites are located at both sides of the VP1 translation start signal. VP1 is translated from the messenger that uses the splice acceptor directly in front of the VP1 translation initiation codon. VP2 and VP3 are translated from messengers that are spliced to the acceptor 3′ of the VP1 ATG. VP2 and VP3 are translated from this messenger by use of an ACG translation start (VP2) or a downstream ATG (VP3). Since all three coding regions are in frame, the capsid proteins share a large domain with an identical amino-acid sequence. VP3 is entirely contained within VP1 and VP2, but the latter two contain additional amino-terminal sequences. Similarly, VP1 contains the entire VP2 protein but carries an additional N-terminal sequence. All three capsid proteins terminate at the same position
8
. The AAV capsid is 20 to 24 nm in diameter
9, 10
and contains approximately 5% VP1, 5% VP2 and 90% VP3. This ratio is believed to reflect the relative abundance of the alternatively spliced messengers and the reduced translation initiation efficiency at the ACG initiation codon for VP2.
During a productive infection, the P5-promoter is activated first and directs the production of the large Rep-proteins, Rep78 and Rep68. These proteins are essential for AAV-replication and trans regulation of viral and cellular genes. The large Rep-proteins activate the P19 and the P40 promoter. In a latent infection, however, Rep78 and Rep68 down regulate expression of the P5 promoter and help to maintain the latency of AAV (for a review see
1
). The smaller Rep-proteins, Rep52 and Rep40, are encoded by transcripts from the P19 promoter and are important for the formation of infectious virus
11
. The P40 promoter is the last promoter to become activated and its activation follows the expression of the late genes of the helper adenovirus. Via alternative splicing, different mRNAs are produced coding for the structural proteins VP1, VP2 en VP3
12
.
Adeno-Associated Virus Vector Technology
The first recombinant AAV vectors were made by replacing sequences from the rep or the cap gene by the sequences of interest
13-15
. Two methods were used to package the recombinant vector. In one method, the vector genome was packaged by co-transfecting into adenovirus infected cells a plasmid containing the vector together with a plasmid containing the missing AAV-gene. In the second method, a plasmid containing the vector was co-transfected with an AAV-genome that was too large to be packaged by an insertion of lambda phage DNA
13-15
. Recombinant virus produced in this way is always contaminated with wild-type AAV (ranging from 10-50% compared to the recombinant titer). This is presumably due to recombination between the two co-transfected plasmids which contain a substantial region of overlap, or by loss of the lambda DNA sequence. The contaminating wild-type AAV causes a further amplification of the rAAV upon infection of a new batch of adenovirus infected cells, leading to higher rAAV-titers but also leading to amplification of the contaminating wild-type AAV
13-15
.
To circumvent the production of wild-type AAV, a packaging plasmid was constructed that contains no overlap with the vector plasmid
7
. With this packaging plasmid, it is possible to generate rAAV virus stocks that are free of detectable amounts of wild-type AAV, while at the same time it enables the production of 0.1 to 1 rAAV particles per cell
7
. This packaging system, or analogous systems derived therefrom, are currently used by most laboratories. Although this is the method of choice at this moment, the method is far from optimal since it cannot easily be scaled up to allow industrial production of rAAV vectors. Plasmid transfections are inherently inefficie
Einerhand Markus Peter Wilhelmus
Valerio Domenico
IntroGene B.V.
Sandals William
Schwartzman Robert A.
TraskBritt
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