Vectors for tissue-specific replication

Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus

Reexamination Certificate

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C435S320100, C435S069100, C435S091400, C435S455000, C536S024100, C514S04400A

Reexamination Certificate

active

06551587

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to targeted gene therapy using recombinant vectors and particularly adenovirus vectors. The invention specifically relates to replication-conditional vectors and methods for using them. Such vectors are able to selectively replicate in a target tissue to provide a therapeutic benefit from the presence of the vector per se or from heterologous gene products expressed from the vector and distributed throughout the tissue. 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. With this vector, therefore, a target tissue can be selectively treated. 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. The invention also relates to cells for producing recombinant replication-conditional vectors useful for targeted gene therapy.
2. Background Art
Targeting Vectors
One of the major goals for therapeutic use of exogenous genes has been cell targeting with high specificity. General approaches have included systemic introduction of DNA, DNA-protein complexes, and liposomes. In situ administration of retroviruses has also been used for cells that are actively replicating.
However, because of the lack of, or significantly low, cell-specificity and inefficient gene transfer, the targeting of desired genes to specific cells in an organism has been a major obstacle for exogenous gene-based therapy. Thus, the use of such genes has been limited.
Tumor cells are among those cell types for which it would be especially desirable to provide a means for exogenous gene targeting. In an embodiment of the present invention, compositions and methods are provide to deliver exogenous genes to tumor cells safely and efficiently.
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.
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. However, as discussed below, the E1a function may be bypassed. Cells may be manipulated to provide E1a-like functions or may naturally contain such functions. The virus may also be manipulated to bypass the functions as described below.
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 transfer vector is that the viral vector generally remains episomal and does not replicate, thus, cell division leads to the eventual loss of the vector from the daughter cells” (Morgan, R. A., et al.,
Annual Review of Biochemistry
62:191-217 (1993)) (emphasis added).
Non-replication of the vector leads not only to eventual loss of the vector without expression in most or all of the target cells but also leads to insufficient expression in the cells that do take up the vector, because copies of the gene whose expression is desired are insufficient for maximum effect. The insufficiency of gene expression is a general limitation of all non-replicating delivery vectors. Thus, it is desirable to introduce a vector that can provide multiple copies of a gene and hence greater amounts of the product of that gene. The present invention overcomes the disadvantages discussed above by providing a tissue-specific, and especially a tumor-specific replicating vector, multiple DNA copies, and thus increased amounts of gene product.
Production of Adenoviral Vectors
Adenoviral v

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