Title:  Coxsackie virus vectors for delivery of nucleic acids encoding antigenic or therapeutic products

United States Patent:  6,323,024

Assignee:  Board of Regents of the University of Nebraska (Lincoln, NE)

Filed:  March 27, 2000

PCT NO:  PCT/US98/04291

102(e) Date:  March 27, 2000

PCT PUB. Date:  September 11, 1998

The present invention is drawn to the use of attenuated Coxsackievirus cardiotropic virus vectors as efficient gene transfer vectors to deliver immunomodulatory or other biologically active proteins and/or antigenic epitopes in transient infections to aid in preventing, ameliorating, and/or ablating infectious viral heart disease and reducing, or ablating entirely, heart transplant rejection. Additionally, other organs or tissues may be targeted with specific picornaviruses. In particular, an attenuated CVB3 viral vector able to express a cytokine is provided. This cytokine-expressing viral vector is able to deliver the cytokine to a target tissue and reduce disease symptoms.

SUMMARY OF THE INVENTION

One object of the present invention is to provide viral vectors for therapeutic or prophylactic use in human disease by delivering nucleic acids encoding antigenic epitopes or specific biologically active gene products, such as (but not limited to) immunomodulatory cytokines, to target cells, tissues or organs in an individual.

Thus, according to one aspect of the invention, a viral vector for delivering a heterologous nucleic acid to a target cell, tissue or organ is provided, which comprises a coxsackievirus genome modified to encode an attenuated coxsackievirus, the genome further comprising at least one cloning site for insertion of at least one expressible heterologous nucleic acid. In a preferred embodiment, the coxsackievirus genome is a coxsackievirus B genome, most preferably a coxsackievirus B3 genome.

In one embodiment of the invention, attenuation of the coxsackievirus is achieved by altering a transcription regulatory region of the genome. Preferably, the transcription regulatory region comprises a 5' untranslated region of the genome. In one embodiment, the 5' untranslated region is replaced with a 5' untranslated region of a non-enterovirus genome selected from the group consisting of poliovirus and echovirus. In another embodiment, a coxsackievirus B3 genome is modified by substituting a C or G for a U at nucleotide position 234 of the genome.

The cloning site of the coxsackievirus vector can be positioned between a coding sequence for a capsid protein and a coding sequence for viral protease. In another embodiment, the cloning site is positioned at the start of the genome's open reading frame, and is constructed such that the inserted expressible heterologous DNA comprises a translation start codon and a 3' sequence recognized by a viral protease.

In one embodiment, the expressible heterologous DNA carried by the coxsackievirus vector of the invention encodes an antigenic product. In another embodiment, it encodes a biologically active product, such as a biologically active protein. Preferably, the protein is a cytokine, such as IL-4 or IL-10. Alternatively, the protein could be another immunomodulatory protein, such as B-7 (B-7-1 or B-7-2).

According to another aspect of the present invention, there is provided a bioengineered virus for the therapeutic delivery of at least one heterologous gene to a target organ or organ system in an individual, comprising a Coxsackievirus B3 (CVB3), wherein said Coxsackievirus B3 is attenuated, and wherein a genome of said CVB3 codes for said at least one heterologous gene. Attenuation of the CVB3 may be accomplished through a transcriptional mechanism. Preferred embodiments include attenuating the virus by substituting a cytosine or guanosine nucleotide for a uracil nucleotide at position nt234 in the genome of the coxsackievirus B3. Another preferred embodiment includes point mutations at positions nt233 and nt236 in the genome of the Coxsackievirus B3, or deletion entirely of nt 233-236.

In addition, the 5' non-translated region of the genome of the Coxsackievirus B3 may be substituted with a 5' non-translated region of a genome from a non-enterovirus to achieve attenuation. In a preferred embodiment, the non-enterovirus is a poliovirus or echovirus.

In most preferred embodiments, the genome of the bioengineered Coxsackievirus B3 includes the basic CVB3/0 genome (as reported by Chapman, N. M., et al, Arch. Virol. 135: 115-130 (1994)), wherein a coding sequence for a heterologous gene is inserted between a capsid protein coding sequence and a viral protease coding region site. Alternatively, a heterologous gene may be inserted at the start of the open reading frame, directly upstream of capsid protein 1A, start with the initiation codon AUG, and end with a sequence recognized by a viral protease. In this preferred embodiment, an immunomodulatory gene or a gene for an antigenic epitope is used. In a more preferred embodiment, cytokine genes are delivered. In a most preferred embodiment, the cytokine is IL-4 or IL-10. Up to seven cytokine genes may be delivered in one vector. Further, both antigenic epitopes and cytokines may be delivered at the same time. Also, a preferred embodiment utilizes sequences for viral proteases P2-A and P3-C.

A further object of the present invention is to provide a method for suppressing an immune response in an individual, comprising the step of administering the bioengineered therapeutic virus containing an immunomodulatory gene to an individual.

An additional object of the present invention is to provide a method for vaccinating an individual, comprising the step of administering the bioengineered therapeutic virus containing a gene for an antigenic epitope to an individual.

Claim 1 of 30 Claims

What is claimed is:

1. A viral vector comprising a coxsackievirus genome modified to encode an attenuated coxsackievirus, said genome further comprising at least one cloning site for insertion of at least one expressible heterologous nucleic acid.

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