Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-08-24
2004-08-24
Wehbe, Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C435S455000, C424S093210, C514S04400A
Reexamination Certificate
active
06780639
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of biotechnology and is related to a new antibiotic inducible/repressible genetic construct or system for improving particle the control of gene therapy or gene immunization. In particular, a system or genetic construct adapted for all types of gene therapy and gene immunization based upon the use of naked DNA or DNA incorporated into various vectors (such as plasmide, adeno-associated viruses, autonomous parvoviruses, retroviruses or adenoviruses or a combination thereof).
BACKGROUND OF THE INVENTION
Various systems comprising naked DNA or DNA incorporated into a suitable vector (plasmid, virus, cationic visicule, . . . ) are used in gene therapy or gene immunization (vaccine). Various publications describe for gene transfer into cells, the use of adeno-associated viruses, which are human defective parvoviruses whose genomes are made of single stranded DNA molecules. Six or five different serotypes have been cloned in prokaryotic plasmide and could be used to derive vectors.
The international patent application PCT/US95/04587 describes gene delivery to adult CNS using AAV vectors.
Humans suffering from Parkinsonism have been treated by striatal implantation of foetal dopaminergic neurons (Lindvall et al., Arch. Neurol. 46:615-631 (1989); Widner et al. New Engl. J. Med. 327: 1556-1563 (1992). Following surgery, the patients exhibited improvement of neurological function. Grafts partially re-establish dopaminergic activity and ameliorate motor functions. However, the success of foetal brain tissue transplantation into impaired area of Parkinson's and Huntington's patients brain is limited by the poor survival of the graft. To ensure maximal viability, the foetal tissue must be freshly harvested prior to transplantation. Recent advances consist of keeping the tissue refrigerated (at 4° C.) for 24 hours without loss of viability. Nevertheless, the coordination between the harvesting of the foetal tissue and the transplantation procedure is still a problem. Furthermore, the amount of foetal tissue available for transplantation is limited for practical and ethical reasons. Foetal tissue is technically difficult to obtain, particularly if multiple donors are needed for each patient. This limits the widespread applicability of foetal tissue transplantation.
The supply of Glial cell line-Derived Neurotrophic Factor (GDNF), a neurotrophic factor for dopaminergic neurons, could promote the protection of rafted cells as well as of remaining host dopaminergic cells. However, since neurotrophic factors can not cross he brain-blood-barrier, they have to be administrated directly in the brain in sustained levels.
The international patent application (PCT)/US96/05814 describes a method of using neurotrophic actors to enhance neuronal survival and promote functional integration of grafted neurons using osmotic umps implanted in the brain. This technique is difficult to implement in the clinics, in particular because of the risk of bacterial contamination.
Improving the survival of the grafted tissue by transfer of genes coding for neurotrophic factors would reduce the amount of tissue needed per patient and make the transplantation therapy available to a greater number of patients.
Stable genetic modification of the graft cells by the means of viral vectors expressing trophic factors could be used to enhance the survival of the grafted tissue.
Genetically-modified foetal mesencephalon fragments or dissociated cell suspensions expressing GDNF could be grafted in order to obtain i) a better survival of the graft (autocrine effect), ii) the protection of host's dopaminergic terminals in the putamen and of a dopaminergic cell bodies in the substantia nigra after retrograde transport of GDNF (paracrine effect).
Furthermore, the combination of autocrine and paracrine effects could result in a better correction of parkinsionnian symptoms by foetal grafts transplants.
Adeno-associated virus is a human defective parvovirus whose genome is a single stranded DNA molecule. Five different serotypes have been cloned in prokaryotic plasmide and could be used to derive vectors.
For efficient replication AAV requires a co-infection with a so-called “helper virus”, usually adenovirus or herpes simplex virus. In the absence of helper virus, AAv can still enter host cells but it stays latent with his genome integrated in the cellular genome. The genome is flanked by 2 inverted terminal repeats (ITRs) which serve as a replication origin.
The double-stranded form of AAV type 2 has been cloned in a pBR322 plasmid allowing the genetic analysis of the virus as well as the development of vectors for gene transfer (Samulski et al. 1982).
It was soon realized the non-coding ITRs are the only elements required in cis for replication and encapsidation of the viral genome (McLaughlin et al., 1987). Accordingly, the vectors derived from AAV only retain the ITRs; the internal coding region is replaced by the desired transgene(s) and regulatory elements (Samulski et al. 1989). To produce recombinant viral particles, a plasmid containing the ITRs flanking the transgene expression cassette is transfected into producer cells in which AAV rep and cap genes as well as necessary helper virus genes are provided either by transfection or by infection.
AAV vectors transduce various types of neurons in the adult rat (McCown et al., 1997, Klein et al., 1998) and monkey (During et al., 1998) as well rodent and human neurons in culture (Du et al., 1996). Human brain slices from epileptic patients could also be transduced by AAV vectors (Freese et al., 1997). AAV vectors were shown to integrate in neurons (Wu et al., 1998).
The international PCT/US95/04587 describes gene delivery to adult CNS using AAV vectors.
However, controllable gene expression is a prerequisite for safe gene therapy or gene immunization in many protocols: for example, erythropoietin level is critical for the treatment of &bgr;-thallaseemia.
In models for Parkinson's disease, the intrastriatal delivery of AAV viral vectors encoding GDNF resulting in long-term overexpression of GDNF effectively protects dopaminergic neurons but also results in side-effects on neighboring normal cells (Kirik et al., 2000). PCT/US94/06734 describes a prokaryotic tetracycline system to produce a genetic switch for achieving control of eukaryotic gene expression. In the native prokaryotic tetracycline system, tetracycline is an effector that induces prokaryotic gene expression by binding to a tetracycline repressor protein. In the absence of tetracycline, the tetracycline repressor binds to a tetracycline operator sequence, which is linked to a promoter and represses transcription. In the presence of tetracycline, the tetracycline repressor binds tetracycline, which binding displaces the repressor from the tetracycline operator sequence , so repression is relieved and transcription can begin.
This tetracycline-controlled activator system is constructed by fusing a tetracycline repressor to a transcription activation domain from a protein that activates transcription in eukaryotic cells. In the absence of tetracycline, the tetracycline-controlled activator (tTa) binds the tetracycline operator sequence which is linked to a promoter and activates transcription. In the presence of tetracycline, the tetracycline-controlled activator binds tetracycline, which binding displaces the activator from the tetracycline operator sequence so activation is ended and transcription is silenced. This is a tetracycline-repressible system.
In a further adaptation, the tetracycline-transactivator is mutated in such a way that it binds the tetracycline operator sequence only when tetracycline binds to the mutant tetracycline transactivator (rtTA). Consequently, in the absence of tetracycline, transcription does not occur. In the presence of tetracycline, transcription can begin. This is a tetracycline-inducible system.
These regulatory systems require that two different expression vectors enter each cell. A first expression vector e
Brotchi Jacques
Chtarto Abdelwahed
Levivier Marc
Peschanski Marc
Tenenbaum Liliane
Knobbe, Martens, Olson & Bear, L L P
Li Janice
Universite Libre de Bruxelles
Wehbe Anne M.
LandOfFree
Antibiotic inducible/repressible genetic construct for gene... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Antibiotic inducible/repressible genetic construct for gene..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antibiotic inducible/repressible genetic construct for gene... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3341613