Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-04-13
2003-01-07
Martinell, James (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S455000, C435S456000
Reexamination Certificate
active
06503888
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the delivery of DNA to and the expression of delivered genes in, cells of the nervous system.
2. Description of the Related Art
The first human gene therapy trial started in September 1990 and involved retrovirally-mediated transfer of the adenosine deaminase (ADA) gene into lymphocytes of patients with severe combined immunodeficiency (SCID). The, favorable results of this trial stimulated further interest in gene therapy resulting in further 67 gene therapy clinical protocols approved by the NIH Recombinant DNA Advisory Committee (RAC) to date. Although the original promise of gene therapy was the development of a curative treatment for simple, single gene diseases, the vast majority of gene therapy trials have been for complex genetic or acquired diseases, such as infectious disease and cancer. A large number of the initial clinical gene transfer studies were not gene therapy but rather gene marking studies. The first type of marking experiments used tumor infiltrating lymphocytes which were transduced in vitro with retroviral vectors prior to infusion into patients with cancer. The second class of gene marking studies involved the attempt to detect residual tumor cells in marrow infused into patients following ablative chemotherapy.
Of the currently approved gene therapy trials, all trials prior to 1992 used retroviral vectors and the diseases targeted included SCID, familial hypercholesteremia and cancer. More recently, gene therapy trials have commenced for AIDS and Hemophilia B, again using retroviral vectors. In addition, adenoviral vectors have recently been approved for cystic fibrosis. The vast majority of these protocols have enrolled very few patients at present and most of the trials are as yet unpublished. The available data however appears promising, for example the expression of the LDL receptor in the liver following ex vivo transduction of resected hepatocytes and its infusion into the portal vein in patients with familial hypercholesteremia has resulted in a 20% drop in plasma cholesterol levels (Randall (1992)
JAMA
269:837-838). It is likely therefore that there will be an exponential growth in gene therapy trials and a large number of medical schools and teaching hospitals are setting up gene therapy centers.
The ability to deliver genes to the nervous system, and to manipulate their expression, may make possible the treatment of numerous neurological disorders. Unfortunately, gene transfer into the central nervous system (CNS) presents several problems including the relative inaccessibility of the brain and the blood-brain-barrier, and that neurons of the postnatal brain are post-mitotic. The standard approach for somatic cell gene transfer, i.e., that of retroviral vectors, is not feasible for the brain, as retrovirally mediated gene transfer requires at least one cell division for integration and expression. A number of new vectors and non-viral methods have therefore been used for gene transfer in the CNS. Although the first studies of gene transfer in the CNS used an ex vivo approach, i.e., the transplantation of retrovirally-transduced cells, more recently several groups have also used an in vivo approach. Investigators have used HSV-1 and adenoviral vectors as well as non-viral methods including cationic lipid mediated transfection (Wolff (1993)
Curr. Opin. Biol
. 3:743-748).
The ex vivo approach is illustrated by a recent study in which oligodendrocytes were retrovirally infected and transplanted into a syngeneic rat model for demyelination (Groves et al (1993)
Nature
362:453-457). In addition to the use of brain cells as vehicles for foreign gene expression in the CNS, non-neuronal cells including fibroblasts and primary muscle cells have also been used (Horrelou et al (1990)
Neuron
5:393-402; Jiao et al (1993)
Nature
362:450-453).
The in vivo approach was initially largely based on the use of the neurotropic Herpes Simplex Virus (HSV-1), however, HSV vectors present several problems, including instability of expression and reversion to wild-type (see below). A more recent development has been the use of adenoviral vectors. Adenoviral vector studies have shown expression of marker genes into the rat brain persisting for two months although expression fell off dramatically (Davidson et al (1993)
Nature Genetics
3:219-2223). In addition to viral vector approaches, other investigators have used direct injection of a cationic liposome:plasmid complex obtaining low level and transient expression of a marker gene (Ono et al (1990)
Neurosci. Lett.
117:259-263).
There have been very few studies using “therapeutic” genes in the CNS. The majority of these have used the ex vivo approach with transduction of fibroblasts and muscle cells with the human tyrosine hydroxylase gene in order to produce L-dopa-secreting cells for use in models of Parkinson's Disease (e.g., Horrelou et al (1990)
Neuron
5:393-402; Jiao et al (1993)
Nature
362:450-453). Of the in vivo approaches, HSV vectors have been used to express &bgr;-glucuronidase (Wolfe et al (1992)
Nature Genetics
1:379-384), glucose transporter (Ho et al (1993) Proc.
Natl. Acad. Sci
. 90:6791-6795) and nerve growth factor (Federoff et al (1992)
Proc. Natl. Acad. Sci
. 89:1636-1640). An adenoviral vector has been used to induce low level transient expression of human &agr;1-antitrypsin (Bajoccchi et al (1993) 3:229-234).
The only clinical studies of gene transfer in the brain followed a report by Culver et al (1992)
Science
256:18550-18522) in which they essentially cured rats which had been intracerebrally implanted with glioma cell lines. They used a retrovirus expressing the HSV-1 thymidine kinase (tk) gene and then subsequently treated with ganciclovir. In 1993, a human protocol for glioblastoma multiforme was approved using the retroviral tk vector—ganciclovir protocol (Oldfield et al (1993)
Human Gene Ther
. 4:39-69).
Herpes Viruses The genome of the herpes simplex virus type-1 (HSV-1) is about 150 kb of linear, double-stranded DNA, featuring about 70 genes. Many viral genes may be deleted without the virus losing its ability to propagate. The “immediately early” (IE) genes are transcribed first. They encode trans-acting factors which regulate expression of other viral genes. The “early” (E) gene products participate in replication of viral DNA. The late genes encode the structural components of the virion as well as proteins which turns on transcription of the IE and E genes or disrupt host cell protein translation.
After viral entry into the nucleus of a neuron, the viral DNA can enter a state of latency, existing as circular episomal elements in the nucleus. While in the latent state, its transcriptional activity is reduced. If the virus does not enter latency, or if it is reactivated, the virus produces numerous infectious particles, which leads rapidly to the death of the neuron. HSV-1 is efficiently transported between synaptically connected neurons, and hence can spread rapidly through the nervous system.
Two types of HSV vectors have been utilized for gene transfer into the nervous system. Recombinant HSV vectors involve the removal of an immediate-early gene within the HSV genome (ICP4, for example), and replacement with the gene of interest. Although removal of this gene prevents replication and spread of the virus within cells which do not complement for the missing HSV protein, all of the other genes within the HSV genome are retained. Replication and spread of such viruses in vivo is thereby limited, but expression of viral genes within infected cells continues. Several of the viral expression products may be directly toxic to the recipient cell, and expression of viral genes within cells expressing MHC antigens can induce harmful immune reactions. In addition, nearly all adults harbor latent herpes simplex viruses within neurons, and the presence of recombinant HSV vectors could result in recombinations which can produce an actively replicating wild-type virus. Alternatively,
During Matthew J.
Kaplitt Michael G.
Klauber & Jackson
Martinell James
The Rockefeller University
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