Assays for promoter operability in central nervous system cells

Details Assays for promoter operability in central nervous system cells Assays for promoter operability in central nervous system cells

1995-06-02

2002-10-01

Priebe, Scott D. (Department: 1632)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007910, C435S455000, C435S320100, C424S093100, C424S093200

Reexamination Certificate

active

06458529

ABSTRACT:

To transfer safely and efficiently therapeutic DNA into the central nervous system, is a formidable challenge in the development of active therapies in brain diseases.
Preliminary investigations have been carried out with a number of vectors, more particularly retroviral vectors and herpes simplex derived vectors. However the usefulness of such gene transfer vehicles has, to date, been limited.
In most cases, retroviral vectors are not useful because they are unable to infect postmitotic cells, including most neural cells (1). Herpes simplex derived vectors infect neural cells but problems of pathogenicity and of stability of gene expression remain unsolved (2,3). In addition Herpes simplex derived vectors have so far proven to have but limited efficacy of expression. In a most recent article (4) the authors refer to the short-term expression reported earlier of HSV-1 derived vectors, because most of the promoters used had only been active during the acute phase of viral infection (less than 10 days post-infection). They disclosed the expression of a b-glucuronidase gene in a cell of the central nervous system under the control of the LAT promoter normally associated with the latency-associated-transcript (LAT) sequence of the virus. But the authors also report that, even though their experiments demonstrated the feasibility of using the LAT promoter for long-term expression of foreign genes in cells of the central nervous system to correct a genetic enzymatic deficiency in infected cells, too few cells had been corrected to alter the disease phenotype. Consequently their vector system needed to be improved to correct sufficient cells for obtaining a clinically significant effect.
The invention aims at obviating such difficulties and at providing most effective vector systems capable of delivering foreign genes and, where appropriate, their transcription products or expression products directly to cells of the central nervous system, particularly to terminally differentiated cells, incapable of proliferation. A more particular object of the invention is also to allow for the wide spreading of such vector systems throughout the neural tissue to be infected, yet while remaining substantially confined thereto.
Still another object of the invention is to produce such vector systems which are sufficiently safe to allow for a study and regulation in vitro of cloned genes in such cells or in test animals, and for therapy, in man or animal, involving the in situ production of a selected expression product, including gene therapy.
The invention is based on the recognition that adenovirus-derived vectors, particularly non-replicative adenovirus vectors, are capable of fulfilling these aims, both in vitro and in vivo. They provide powerful delivery systems of genes into the cells of the central nervous system, more particularly brain cells. They are characterized by a degree of infectivity of sufficient magnitude to allow for the infection of considerable populations of cells The biological experiments disclosed hereafter demonstrate the capability of adenovirus derived vectors (or adenoviral vectors) of efficiently infecting nerve cells, particularly neurons, both in vitro and in vivo.
Thus the invention provides a process for the production of a recombinant vector useful in a method comprising causing the transcription product or the expression product of a nucleotide sequence coding for a selected polypeptide to be targeted or produced in cells of the central nervous system, e.g. brain cells, particularly neural, glial or ependymal cells, wherein said recombinant vector is an adenoviral vector which comprises at least part of the genome of an adenovirus including those regions of that genome which provide the genetic information required by that adenovirus to penetrate into cells normally infectable by it, said nucleotide sequence being inserted in said genome part, under the control of a promoter either present or also inserted within said adenoviral vector, and said promoter being operative in said cells.
Thus the invention is more particularly related to the use of an adenovirus-derived vector for the expression of a selected nucleotide in the cells of the central nervous system.
The invention also provides a recombinant DNA vector characterized in that it is capable of directing the expression and/or transcription of a selected nucleotide sequence in the cells of the central nervous system and in that it comprises (i) at least part of the genome of an adenovirus, including the regions required for that adenovirus to penetrate into the cells of the central nervous system, and (ii) said selected nucleotide sequence under the control of a promoter operative in said cells.
The powerful capability of adenoviral vectors of transferring gene fragments in vivo into quiescent neural cells is illustrated by the experiments reported hereafter, which were carried out with an adenovirus vector carrying the
E. Coli
lac Z gene or the human tyrosine hydroxylase gene, in neural cells of adult rats. A large number of neural cells (including neurons, astrocytes, microglia and ependymal cells) expressed these transgenes at least 60 days after inoculation of various brain areas. Injecting up to 3×10
5
pfu in 10 &mgr;l did not result in any detectable cytopathic effects, which were only observed for the highest titers of infection (>10
7
pfu/10 &mgr;l) and were most likely associated with a massive endocytosis of viral particles in neural cells close to the injection site.
Moreover the genomes of adenoviruses can be manipulated to accommodate foreign genes of 7.5 kb in length or more. It has a large host range, a low pathogenicity in man, and high titers of the virus can be obtained (5).
It will be readily appreciated that these results strongly support the presumption that adenovirus offers, as a vector, a new and remarkable tool to genetically modify quiescent cells of the nervous system, because of its great efficacy of infection, long term expression, wide host range and low toxicity. Thus, adenovirus should be instrumental in the study of the function of cloned gene products in their physiological and anatomical context. The ability to infect the hippocampus (as this will be shown later) is of great interest to study integrated phenomena such as long-term potentiation, in animals.
Moreover the adenovirus clearly appears as an efficient means to transfer foreign genes into the brain with a therapeutic goal. Adenovirus vectors have great potential for gene therapy of nervous system diseases, such as the local delivery of growth factors or neurotransmitters for degenerative diseases and, more generally, to replace defective genes in appropriate cells. Relatively low titers of adenovirus vectors can efficiently transfer foreign genes into a significant number of brain cells without triggerring pathological effects. Subject to optimization of the doses of recombinant adenoviral vectors containing a foreign gene to be delivered into brain cells, they open new avenues in the treatment of many genetic and acquired neurological diseases, consequently, an alternative to drug treatment or brain transplantation of fetal tissues.
Adenovires, particularly adenoviruses of type 2 or 5 (Ad2 or Ad5) are particularly preferred. They are relatively stable, can be cultured easily and rapidly (viral cycle of about 30 hours) and provide high titers: up to 10
4
-10
5
plaque forming units (p.f.u.) per infected cell. They are not oncogenic. The complete sequence of their viral genome has been established (6) and its molecular biology has been studied extensively. Finally several mutants, particularly deletion mutants, have been obtained, which makes it possible to insert fragments of large size therein (7).
Preferably the recombinant vectors for use in this invention are defective adenoviruses, whose genomes no longer contain those nucleotides sequences required for the virus replication in cells (other than brain cells) normally infectable by it. More particularly, they are free of the E1 region, including

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