Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1997-11-19
2003-10-28
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S069100, C435S091400, C435S320100, C435S455000, C514S04400A, C424S093200
Reexamination Certificate
active
06638762
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to cell-specific expression vectors. It particularly relates to targeted gene therapy using recombinant expression vectors and particularly adenovirus vectors. The invention specifically relates to modulatable replication-conditional expression vectors and methods for using them. Such vectors are able to selectively replicate in a target cell or tissue to provide a therapeutic benefit in a tissue from the presence of the vector per se or from one or more heterologous gene products expressed from the vector and distributed throughout the tissue, and which vectors are designed so that replication and gene expression from the vector can be modulated.
In such vectors, a gene essential for replication is placed under the control of a heterologous tissue-specific transcriptional regulatory sequence. Thus, replication is conditioned on the presence of a factor(s) that induces transcription or the absence of a factor(s) that inhibits transcription of the gene by means of the transcriptional regulatory sequence.
Preferred vectors contain a heterologous gene that produces a product that increases or inhibits viral replication. Such genes are useful for modulating viral replication and thus also for modulating expression of genes in the vector. With these vectors, therefore, the vector can be expressed in a desirable cell, target tissue can be selectively treated, and replication and expression modulated.
The invention also relates to cells and/or methods to produce multiple heterologous gene products in high quantity from essentially one promoter element.
The invention also relates to methods of using the vectors to screen a tissue for the presence or absence of transcriptional regulatory functions that permit vector replication by means of the transcriptional regulatory sequence.
2. Background Art
Targeting Vectors
The introduction of exogenous genes into cells in vitro or in vivo, systemically or in situ, has been of limited use for compositions in which it would be disadvantageous for non-target cells to take up the exogenous gene. One strategy to overcome this problem is to develop administration procedures or vectors that target a specific cell-type. Using systemic administration, attempts have been made to direct exogenous genes to myocytes and muscle cells by direct injection of DNA, to direct the exogenous DNA to hepatocytes using DNA-protein complexes, and to endothelial cells using liposomes.
Using in situ administration, retroviral replication functions have been utilized to target cells that are actively replicating.
Thus far, the ability to target cells has been limited, however, by the lack of cell-type specificity and low gene transfer efficiencies. The limited ability to target an exogenous gene to diseased cells in an organism, while avoiding (eliminating) uptake of the gene by normal, untargeted cells, particularly has been an obstacle to developing effective gene-transfer-based therapies for diseases in animals and humans.
One especially difficult challenge is targeting tumor cells. Many seemingly promising strategies for these cells, moreover, are limited to one or a few cell-types.
The present invention, in one aspect, provides a way to deliver an exogenous gene efficiently, with high distribution in a tumor and in a controlled manner.
Adenoviruses Generally
Adenoviruses are nonenveloped, regular icosohedrons. The protein coat (capsid) is composed of 252 capsomeres of which 240 are hexons and 12 are pentons. Most of the detailed structural studies of the adenovirus polypeptides have been done for adenovirus types 2 and 5. The viral DNA is 23.85×10
6
daltons for adenovirus 2 and varies slightly in size depending on serotype. The DNA has inverted terminal repeats and the length of these varies with the serotype.
The replicative cycle is divided into early (E) and late (L) phases. The late phase defines the onset of viral DNA replication. Adenovirus structural proteins are generally synthesized during the late phase. Following adenovirus infection, host DNA and protein synthesis is inhibited in cells infected with most serotypes. The adenovirus lytic cycle with adenovirus 2 and adenovirus 5 is very efficient and results in approximately 10,000 virions per infected cell along with the synthesis of excess viral protein and DNA that is not incorporated into the virion. Early adenovirus transcription is a complicated sequence of interrelated biochemical events, but it entails essentially the synthesis of viral RNAs prior to the onset of viral DNA replication.
The organization of the adenovirus genome is similar in all of the adenovirus groups and specific functions are generally positioned at identical locations for each serotype studied. Early cytoplasmic messenger RNAs are complementary to four defined, noncontiguous regions on the viral DNA. These regions are designated (E1-E4). The early transcripts have been classified into an array of immediate early (E1a), delayed early (E1b, E2a, E2b, E3 and E4), and intermediate (IVa2.IX) regions.
E1a is a transactivator of multiple gene products in adenovirus through activation of the E1b, E2, E3 and E4 promoters. The E1a region is involved in transcriptional transactivation of viral and cellular genes as well as transcriptional repression of other sequences. The E1a gene exerts an important control function on all of the other early adenovirus messenger RNAs. In normal tissues, in order to transcribe regions E1b, E2a, E2b, E3, or E4 efficiently, active E1a product is required.
The E1b region is required for the normal progression of viral events late in infection. The E1b product acts in the host nucleus. Mutants generated within the E1b sequences exhibit diminished late viral mRNA accumulation as well as impairment in the inhibition of host cellular transport normally observed late in adenovirus infection (Berkner, K. L.,
Biotechniques
6:616-629 (1988)). E1b is required for altering functions of the host cell such that processing and transport are shifted in favor of viral late gene products. These products then result in viral packaging and release of virions. E1b produces a 19 kD protein that prevents apoptosis. E1b also produces a 55 kD protein that binds to p53.
For a complete review on adenoviruses and their replication, see Horwitz, M. S.,
Virology
2d
ed
., Fields, B. N., eds., Raven Press Limited, New York (1990), Chapter 60, pp. 1679-1721.
Adenovirus as Recombinant Delivery Vehicle
Adenovirus provides advantages as a vector for adequate gene delivery for the following reasons. It is a double stranded DNA nonenveloped virus with tropism for the human respiratory system and gastrointestinal tract. It causes a mild flu-like disease. Adenoviral vectors enter cells by receptor mediated endocytosis. The large (36 kilobase) genome allows for the removal of genes essential for replication and nonessential regions so that foreign DNA may be inserted and expressed from the viral genome. Adenoviruses infect a wide variety of cell types in vivo and in vitro. Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenovirus vectors rarely integrate into the host chromosome; the adenovirus genome remains as an extrachromosomal element in the cellular nucleus. There is no association of human malignancy with adenovirus infection; attenuated strains have been developed and have been used in humans for live vaccines.
For a more detailed discussion of the use of adenovirus vectors for gene therapy, see Berkner, K. L.,
Biotechniques
6:616-629 (1988); Trapnell, B. C.,
Advanced Drug Delivery Reviews
12:185-199 (1993).
Adenovirus vectors are generally deleted in the E1 region of the virus. The E1 region may then be substituted with the DNA sequences of interest. It was pointed out in a recent article on human gene therapy, however, that “the main disadvantage in the use of adenovirus as a gene t
Chang Yung-Nien
Hallenbeck Paul L.
Hay Carl M.
Stewart David A.
Genetic Therapy, Inc.
Golightly Douglas A.
Meigs J. Timothy
Nguyen Dave T.
Savitsky Thomas R.
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