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Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof

A technology of recombinant nucleic acid, recombinant bacteria, applied in the direction of chemical instruments and methods, bacteria, bacterial peptides, etc., can solve the problem that the sensor or cancer cells cannot be guaranteed to be attenuated, attenuated or inactivated pathogens or cells are unsuccessful, etc. question

Inactive Publication Date: 2007-02-28
ADURO BIOTECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Heterologous antigen delivery is particularly advantageous for the treatment or prevention of diseases or conditions caused, inter alia, by toxic or lethal sources such as cancer or pathogens (e.g., HIV or hepatitis B), where injection of natural sensors or cancer cells affects the affected organisms are potentially harmful and administration of attenuated or inactivated pathogens or cells has proven unsuccessful in eliciting an effective immune response, or where adequate attenuation of the sensor or cancer cells cannot be assured with certainty

Method used

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  • Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof
  • Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof
  • Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof

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Embodiment 28

[0369] Alternatively, other known intergenic regions or synthetic sequences can be used to construct polycistronic expression cassettes for use in Listeria or other bacteria. The construction of intergenic regions leading to substantial secondary RNA structure should be prevented to avoid unnecessary transcription termination through rho-independent mechanisms.

[0370] Importantly, if secretion of any or all translated proteins expressed from polycistronic messages is required, the signal peptide must be functionally linked to each coding region. In some embodiments, these signal peptides are different from each other.

[0371] Therefore, in some embodiments, the expression cassettes described herein for use in Listeria or other bacteria are polycistronic (e.g., bicistronic). Two or more polypeptides as discrete polypeptides are encoded by a bicistronic or polycistronic expression cassette. In some embodiments, the bicistronic or polycistronic expression cassette includes an inte...

Embodiment 1

[0540] Example 1. Preparation of exemplary mutant Listeria strains

[0541] The Listeria strain is derived from 10403S (Bishop et al., J. Immunol. 139:2005 (1987)). A recognized method was used to generate Listeria strains with in-frame deletions of the indicated genes by SOE-PCR and allelic exchange (Camilli, et al., Mol. Microbiol. 8:143 (1993)). The mutant strain LLO L461T (DP-L4017) is described in Glomski et al., J. Cell. Biol. 156: 1029 (2002), which is incorporated herein by reference. The actA-mutant strain (DP-L4029) is the DP-L3078 strain described in Skoble et al., J. of Cell Biology, 150: 527-537 (2000), which is hereby incorporated as a reference in its entirety, and its prophage has self-healed . (Prophage self-healing is described in (Lauer et al., J. Bacteriol. 184: 4177 (2002); US Patent Publication No. 2003 / 0203472)). actA - uvrAB - The construction of the strain is described in U.S. Provisional Application 60 / 446,051, filed on February 6, 2003, such as L4...

Embodiment 2

[0543] Example 2. Construction of Listeria strains expressing AH1 / OVA or AH1-A5 / OVA

[0544] A mutant Listeria strain expressing a truncated form of the model antigen ovalbumin (OVA) was prepared, and an immunodominant epitope called AH1 (SPSYVYHQF (SEQ ID NO) from mouse colorectal cancer (CT26) : 72)); and altered epitopes AH1-A5 (SPSYAYHQF (SEQ ID NO: 73); Slansky et al., Immunity, 13: 529-538 (2000)). The pPL2 integration vector (Lauer et al., J Bacteriol. 184:4177 (2002); U.S. Patent Publication No. 2003 / 0203472) was used to generate single copies of OVA and AH1- containing a harmless site integrated into the Listeria genome. A5 / OVA recombinant Listeria strain.

[0545] A. Construction of Listeria expressing OVA (DP-L4056)

[0546] First, an antigen expression cassette (pPL2 / LLO-OVA) composed of a hemolysin-deficient LLO fused with a truncated OVA and contained in the pPL2 integration vector was prepared. By attaching tRNA to the PSA (phage from ScottA) Arg -attBB' introduces ...

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Abstract

The present invention provides recombinant nucleic acid molecules, expression cassettes and vectors for expressing polypeptides, including heterologous polypeptides such as antigens, in bacteria. Some recombinant nucleic acid molecules, expression cassettes and vectors include codon-optimized sequences encoding polypeptides and / or signal peptides. Some recombinant nucleic acid molecules, expression cassettes and expression vectors include sequences encoding non-Listerial and / or non-secAl signal peptides for secreted polypeptides. The present invention also provides bacteria comprising nucleic acid molecules, expression cassettes and expression vectors, and compositions such as vaccines comprising the bacteria. Methods of making and using the bacteria, recombinant nucleic acid molecules and expression cassettes are also provided.

Description

[0001] Cross reference of related applications [0002] According to 35 U.S. C.§119(e), this application claims priority rights for each of the following U.S. provisional applications, the disclosure of each of which is hereby incorporated by reference: U.S. Provisional Application Serial No. 60 / 616,705, the title of the invention is "Bacterial expression cassettes, bacterial vaccine compositions and methods for their use", Thomas W. Dubensky, Jr, etc., filed on October 6, 2004 (Docket No.282173003923); U.S. Provisional Application Series No.60 / 615,287, Title of Invention "Bacterial expression cassettes, bacterial vaccine compositions and methods of use", Thomas W. Dubensky, Jr, etc., filed on October 1, 2004 (Docket No.282173003922); U.S. Provisional Application Series No.60 / 599,377, 2004 Filed on August 5; U.S. Provisional Application Series No. 60 / 556,744, filed on March 26, 2004; U.S. Provisional Application Series No. 60 / 541,515, filed on February 2, 2004; and U....

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K39/02A61P31/00A61K48/00C07H21/04C07K14/195C12N1/21C12N15/74
CPCC07K2319/02A61K2039/523A61K39/00C07K14/195
Inventor T·W·小杜本斯基D·A·波特努瓦W·S·小勒基特D·N·库克
Owner ADURO BIOTECH
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