Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-09-27
2002-06-04
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S455000, C435S457000, C435S235100, C435S243000, C435S247000, C435S250000, C435S260000, C424S093200, C424S093100, C424S093600
Reexamination Certificate
active
06399385
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the field of gene delivery into host cells, and more particularly, to gene delivery via viral vectors.
BACKGROUND OF THE INVENTION
Viral-mediated gene delivery has been described for delivery of therapeutic genes to patients. One limitation of currently known methods is the generation of neutralizing antibodies (NAB) by the patients immune response against viral capsids, which prohibit significant levels of gene expression upon readministration. Thus, methods of gene delivery which circumvent these immune responses are needed.
Covalent modification of proteins and enzymes with functionalized poly(ethylene) glycol (PEG) has been studied. PEG is an uncharged, hydrophilic, linear polymer that is non-immunogenic and has a very low order of toxicity. Recently, O'Riordan et al have developed a process to covalently link various polyethylene glycols to the capsid proteins of adenovirus [O'Riordan et al,
Hu Gene Therapy
, 10: 1349-1358 (1999)]. However, this method requires incubation for a period of 20 to 24 hours.
What is needed are viral vectors which avoid the limitations of current constructs for re-administration, as well as rapid methods for generating high levels of such constructs.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for conjugating a recombinant virus with polyethylene glycol to enhance transduction efficiency thereof. This method involves the steps of reacting activated PEG and a recombinant virus at room temperature for about 15 minutes to about 2 hours; and stopping the reaction, thereby obtaining PEG-conjugated virus. Most suitably, the activated PEG and the recombinant virus are reacted at a ratio of about 10:1 polyethylene glycol to virus. Desirably, the recombinant virus is present at a concentration of about 1×10
10
to about 1×10
15
particles per ml of reaction solution.
In another aspect, the invention provides a PEG-conjugated virus prepared according to the method of the invention.
In yet another aspect, the invention provides a method for increasing transduction efficiency of a recombinant virus which involves delivering a modified recombinant virus according to the invention to host cells.
In still another aspect, the invention provides a method for re-administration of a molecule to a selected host cell via a viral vector. This method involves the steps of contacting the host cell with a PEG-modified virus according to the invention, wherein said virus comprises a molecule for delivery to a host cell; and contacting the host cells with a recombinant virus comprising the molecule.
In yet a further aspect, the invention provides a composition useful for delivery of a selected molecule to host cells. The composition contains a PEG-conjugated virus prepared according to the method of the invention and a physiologically acceptable carrier.
In still a further aspect, the invention provides a composition that enhances the physical stability of viral vectors. This composition contains a recombinant viral vector comprising a molecule for delivery to host cells, sucrose, and mannitol, wherein the ratio of sucrose to mannitol is about 1 to about 1. Desirably, the composition is lyophilized to a final moisture content of about 1.2% to about 1.7%.
Other aspects and advantages of the invention will be apparent from the following detailed description of the invention.
REFERENCES:
patent: WO 98/39465 (1998-09-01), None
patent: WO 98/39467 (1998-09-01), None
patent: WO 98/44143 (1998-10-01), None
patent: WO 00/29024 (2000-05-01), None
patent: WO-95/106-01 (2001-04-01), None
Maria A. Croyle et al, Development of a Rapid Method for the PEGylation of Adenoviruses with Enhanced Transduction and Improved Stability under Harsh Storage Conditions, Human Gene Therapy 11:1713-1722, Aug. 10, 2000.*
Francis et al., “PEGylation of Cytokines and Other Therapeutic Proteins and Peptides:the Importance of Biological Optimisation of Coupling Techniques”.International Journal of Hematology, 68:1-18 (Jul. 1998).
Kita et al., “Characterization of a Polyethylene Glycol Conjugate of Recombinant Human Interferon”,Drug Design and Delivery, 6:157-167 (Sep. 1990).
Peracchia et al., “Pegylated Nanoparticle from a Novel Methoxpolyethylene Glycol Cyanoacrylate-Hexadecyl Cyanoacrylate Amiphiphilic Copolymer”,Pharmaceutical Research, 15(4):550 (Apr. 1998).
Delgado et al., “The Uses and Properties of PEG-Linked Proteins”,Critical Review in Therapeutic Drug Carrier Systems, 9(3,4):249-304 (1992).
Francis et al., “Polyethylene Glycol Modification: Relevance of Improved Methodology to Tumour Targeting”, 3:321-340 (1996).
Richter et al., “Antibodies Against Polyethylene Glycol Produced in Animals by Immunization with Monomethoxy Polyethylene Glycol Modified Proteins”,Int. Archs Allergy Appl. Immun., 70:124-131 (1983).
O'Riordan et al., “PEGylation of Adenorvirus with Retention of Infectivity and Protection from Neutralizing Antibody in Vitro and in Vivo”,Human Gene Therapy, 10:1349-1359 (May 20, 1999).
Croyle et al., “Stealth Viruses as Efficient Vectors for Gene Therapy”,1999 AAPS Annual Meeting Abstracts Online, vol. 1, issue 4, Abstract#4110, published Sep. 29, 1999.
M. A. Croyle et al., “Development of Novel Formulations That Enhance Adenoviral-Mediated Gene Expression in the Lungin Vitroandin Vivo”,Molecular Therapy, vol. 4, No. 1, pp. 22-28 (Jul. 2001).
D. Fisher et al., “Factors with Impact on the Success of Protein and Virus Pegylation”,Polymer Preprints, 41(1), pp. 1012-1013 (Mar. 2000).
M. A. Croyle et al., “Beta Cyclodextrins Enhance Adenoviral-Mediated Gene Delivery to the Intestine”,Pharmaceutical Research, vol. 15, No. 9, pp. 1348-1355 (1998).
T. Ohsawa et al., “Enhancement of Adenovirus-Medicated Gene Transfer into Dermal FibroblastsIn vitroandIn vivoby Polyethylene Glycol 6000”,Journal of Dermatology, 27: 244-251 (Apr. 2000).
M. A. Croyle et al., “Development of a Rapid Method for the PEGylation of Adenoviruses with Enhanced Transaction and Improved Stability under Harsh Storage Conditions”,Human Gene Therapy, 11: 1713-1722 (Aug. 10, 2000).
M. A. Croyle et al., “Stealth Adenoviruses Blunt Cell-Meditated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung”,Journal of Virology, vol. 75, No. 10, pp. 4792-4801 (May 2001).
Croyle Maria
Wilson James M.
Guzo David
Howson and Howson
The Trustees of the University of Pennsylvania
LandOfFree
Methods for rapid PEG-modification of viral vectors,... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for rapid PEG-modification of viral vectors,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for rapid PEG-modification of viral vectors,... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2892759