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Humanized Viral Vectors and Methods of Use Thereof

a technology of humanized viral vectors and vectors, applied in the field of gene therapy and molecular biology, to achieve the effect of reducing antibody mediated clearance and immune responses, and improving efficiency and consistency

Inactive Publication Date: 2010-04-22
THE CHILDRENS HOSPITAL OF PHILADELPHIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]The present inventor has developed a viral vector gene delivery system wherein the viral vector comprises human proteins affixed to the capsid surface which are recognized as “self” proteins by the human immune system. Coating the capsid with human proteins masks the antigenic viral epitopes, precluding recognition by antibodies and thereby reducing antibody mediated clearance and immune responses. Accordingly, delivery of the transgene in the present system can occur in the presence of pre-existing antibodies, and is predicted to occur with higher efficiency and consistency as the viral vector is not cleared by antibody mediated mechanisms. Additionally, the humanized viral vectors may be administered multiple times because of their ability to evade recognition by antibodies.

Problems solved by technology

However, pre-existing immunity to AAV2, and adaptive immune responses to the non-human components (e.g., viral capsid proteins) of AAV-based gene transfer vectors remains a barrier to achieving consistent and efficient gene transfer and long term expression of therapeutic genes in humans.

Method used

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  • Humanized Viral Vectors and Methods of Use Thereof
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  • Humanized Viral Vectors and Methods of Use Thereof

Examples

Experimental program
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Effect test

example 1

[0083]In the present example, the generation of AAV vectors that are covalently coated with HSA such that most or all antibody binding sites are masked is described.

[0084]Covalent cross-linking of vector and HSA. Appropriate cross linking agents have been developed and described to achieve attachment of two different proteins, and methods of use of such agents is well known to the skilled artisan. For example, certain hetero-bifunctional cross linking reagents could be used to form a covalent linkage between Cys34 of HSA with lysine residues that are present on the surface of purified AAV2. Heterobifunctional cross linking agents and methods of using the same are commercially available from Pierce. Covalent crosslinking of HSA to rAAV2 is advantageous in that the complexes, once formed, are stable.

[0085]AAV2-LacZ preparation and titering. Recombinant AAV2 expressing the LacZ under the control of the CMV promoter was generated using helper virus free transient transfection of HEK293 ...

example 2

[0092]In this example, the generation of AAV vectors that are modified with an AAV binding ligand such that most or all antibody binding sites are masked is described.

[0093]Covalent attachment of an AAV2 binding ligand, such as heparin to HSA is performed. HSA can be modified by attaching a ligand that is known to bind to the surface of AAV2 vectors (e.g., heparin). It is known that AAV2 has binding sites for heparin (60 sites per AAV2 particle). Thus, covalent attachment of heparin to purified HSA can be achieved such that one HSA molecule has one attached heparin molecule. It is most preferable that only one heparin molecule be attached to each HSA molecule, because attachment of 2 or more heparin molecules per HSA would lead to HSA derivatives that would be expected to bind more than one vector particle and thereby lead to vector aggregation. Therefore attachment of heparin to a unique site (e.g. Cys34) on HSA provides a suitable mono-heparin HSA derivative to generate the huAAV2...

example 3

[0106]In an alternative approach, modification of the vector surface by attaching small ligands to the vector surface that are known to bind HSA may be employed. AAV2 vectors derivatized with such ligands could be mixed with HSA, which would then bind in a non-covalent manner. For example, it is known that HSA has a high affinity binding site for certain fatty acids (reviewed in Peters (1996)). Fatty acids could be covalently attached to the surface of AAV2 vectors, and the vectors then incubated with purified HSA under conditions to allow non-covalent binding of HSA to the fatty acids molecules. For example, acylation of the epsilon amino groups of lysine residues to attach long fatty acyl residues to proteins has been described (Plou et al. (1994)). To make huAAV2 vectors using this strategy, long chain fatty acids (eg. palmitoyl chloride) or other relevant fatty acid like molecule could be attached covalently to some (or all) of the approximately 420 Lys residues that are exposed...

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Abstract

The present invention provides humanized viral vectors and methods of use thereof for delivery of transgenes or therapeutic nucleic acids to human subjects. Humanized viral vectors are modified from known viral vectors such as those based on AAV by coating their surface with a human protein such as human serum albumin and optionally a lipid coating or formulation, so that the foreign or non-human nature of the vector is masked. The coating is performed in a manner that reduces or prevents binding of antibodies to the vector surface, thereby reducing or preventing antibody-mediated clearance of vector, but still allowing the vector to transduce target cells and achieve therapeutic gene transfer. Such humanized vectors therefore evade pre-existing immune surveillance, reduce immune responses, and achieve therapeutic gene transfer in the presence of pre-existing antibodies to the viral vector.

Description

[0001]This application is a §365 (c) continuation in part application of PCT / US2008 / 060654 filed 17 Apr. 2008, which in turn claims priority to U.S. Provisional Application 60 / 912,193 filed Apr. 17, 2007, the entire contents of each being incorporated herein by reference as though set forth in full.FIELD OF THE INVENTION[0002]This application relates to the fields of gene therapy and molecular biology. More specifically, this invention provides humanized viral vectors which evade immune surveillance, thereby providing for more efficient and predictable expression of therapeutic polynucleotides and polypeptides, greater duration of expression, and the capacity for re-administration in the presence of existing anti-vector immunity. Preferably, the viral vector is an adeno-associated virus vector.BACKGROUND OF THE INVENTION[0003]Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Ea...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N15/00C12N15/86A61P7/00
CPCC07K14/765C12N15/86C12N2810/85C12N2750/14145C12N2750/14143A61K38/4846C12N9/644C12Y304/21022A61P7/00A61K48/00C12N15/8645A61K47/12A61K47/42C12N7/00
Inventor WRIGHT, JOHN FRASER
Owner THE CHILDRENS HOSPITAL OF PHILADELPHIA
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