WO2010015079A1 - Optimized promoter sequence - Google Patents

Optimized promoter sequence Download PDF

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Publication number
WO2010015079A1
WO2010015079A1 PCT/CA2009/001094 CA2009001094W WO2010015079A1 WO 2010015079 A1 WO2010015079 A1 WO 2010015079A1 CA 2009001094 W CA2009001094 W CA 2009001094W WO 2010015079 A1 WO2010015079 A1 WO 2010015079A1
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seq
promoter
set forth
sequence
nucleotide sequence
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PCT/CA2009/001094
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French (fr)
Inventor
Gary Kobinger
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Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health
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Application filed by Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health filed Critical Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health
Priority to US13/057,529 priority Critical patent/US8663981B2/en
Priority to JP2011521414A priority patent/JP5670330B2/en
Priority to NZ591439A priority patent/NZ591439A/en
Priority to EP09804421.7A priority patent/EP2318537B1/en
Priority to AU2009279334A priority patent/AU2009279334B2/en
Priority to CA2733104A priority patent/CA2733104C/en
Publication of WO2010015079A1 publication Critical patent/WO2010015079A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/14011—Filoviridae
    • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
    • C12N2760/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/14011—Filoviridae
    • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
    • C12N2760/14134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription

Definitions

  • the Kozak consensus sequence or Kozak sequence plays a major role in the initiation of translation and has the consensus gcc ⁇ A/G)ccAUGG.
  • the CAG promoter comprises a hybrid CMV enhancer coupled to a modified chicken ⁇ -actin promoter.
  • nucleotide sequence encoding a CAG promoter sequence as set forth in SEQ ID No. 1.
  • nucleotide sequence comprising the CAG promoter sequence as set forth in SEQ ID No. 1 operably linked to a nucleic acid sequence comprising a Kozak sequence and a nucleic acid sequence encoding a codon-optimized Ebola virus glycoprotein as set forth in SEQ ID No. 2.
  • a purified or isolated functional promoter element having at least 70% or more identity with the nucleotide sequence as set forth in SEQ ID No. 1.
  • Figure 3 Bar graphs showing expression intensity of EGFP reporter gene in transfected HEK 293T cells as determined by FACS. Portions of the 5' untranslated region of pCAGGS downstream of the CAG promoter were systematically removed generating pCAGGS ⁇ 764-eGFP, pCAGGS ⁇ 829-eGFP and pCAGGS ⁇ 947-eGFP. Assays were performed in triplicate and repeated twice, the data shown is from one experiment. Error bars represent the standard deviation of the data. * p ⁇ 0.001.
  • FIG. 4 shows the construction of pCAGGS ⁇ 829.
  • the CAG promoter of the highly efficient expression plasmid pCAGGS is comprised of a CMV-[E enhancer coupled with a modified chicken-beta actin promoter.
  • a minimal promoter sequence was identified following a deletion of 829 base pairs in the intron region using restriction enzyme Eco47ll! and Xbal, we termed this vector pcag-alpha.
  • FIG. 5 shows the construction of pCAGGS ⁇ 764.
  • the CAG promoter of the highly efficient expression plasmid pCAGGS is comprised of a CMV-IE enhancer coupled with a modified chicken-beta actin promoter.
  • PspOMl and Xbai The cohesive ends were filled in and ligated together using Klenow fragment and T4 DNA ligase.
  • FIG. 6 shows the construction of pCAGGS ⁇ 947.
  • the CAG promoter of the highly efficient expression plasmid pCAGGS is comprised of a CMV-IE enhancer coupled with a modified chicken-beta actin promoter.
  • the cohesive ends were filled in and ligated together using Klenow fragment and T4 DNA ligase
  • Described herein is an expression cassette comprising an optimized CAG promoter sequence (SEQ ID No. 1 ), shown in Figure 1.
  • the promoter sequence was generated by deleting 829 base pairs using restriction enyzmes Eco47!ll and Xbal, filling the ends with Klenow and then religated the vector carrying the CAG promoter sequence with T4 Ligase, as shown in Figure 4.
  • this deletion resulted in a promoter element having greater efficiency compared to a 'wild type 1 construct and other constructs containing a larger deletion.
  • Figure 5 shows the construction of pCAGGS ⁇ 764 in which 764 base pairs in the intron region were deleted using restriction enzymes PspOM! and Xbal.
  • Figure 6 shows the construction of pCAGGS ⁇ 947 in which 947 base pairs in the intron region were deleted using restriction enzymes Eco47ll! and Acc65l. In both cases, the cohesive ends were filled in and figated together using Klenow fragment and T4 DNA ligase
  • such a promoter element can be used to drive high level expression of any gene of interest.
  • Figure 3 provides considerable information useful for the construction of similar promoter cassettes. Specifically, one of skill in the art having learned that an approximately 829 base pair deletion produces a promoter having increased expression whereas the 949 base pair deletion negates this increased expression can use this information together with methods known in the art to produce related promoter cassettes without undue experimentation.
  • one of skill in the art could easily construct a series of nested deletions using any of a variety of means known in the art such as for example methods allowing progressive deletion such as nuclease-based methods, restriction enzyme digests, sub-cloning and the like to delineate the exact location of the one or more 'negative' element(s) contained within the 829 base pair deletion fragment responsible for repression of transcriptional expression as well as the location of the one or more 'positive' element(s) located between the end-points of the 829 base pair deletion fragment and the 949 base pair deletion fragment.
  • progressive deletion such as nuclease-based methods, restriction enzyme digests, sub-cloning and the like to delineate the exact location of the one or more 'negative' element(s) contained within the 829 base pair deletion fragment responsible for repression of transcriptional expression as well as the location of the one or more 'positive' element(s) located between the end-points of the 829 base pair deletion fragment and the 949 base pair deletion fragment.
  • constructs such as those described above which are functionally similar, function that is, produce a similar but not necessarily identical level of expression, for example, a promoter cassette having 10%, 20%, 25%, 35% 45%, 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression, preferably 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression when compared to the promoter cassette described herein, wherein both constructs are transformed or transfected into the same or a similar cell line and expression is detected under similar conditions) to applicant's construct.
  • nucleotide sequence comprising a Kozak sequence and a nucleotide sequence encoding an Ebola virus glycoprotein. Shown in Figure 2 is such a sequence wherein the codons encoding the Ebola virus glycoprotein have been selected so as to optimize translational efficiency of the Ebola virus glycoprotein in for example HEK 293T cells (SEQ ID No. 2).
  • the expression cassette comprises a nucleotide sequence encoding the CAG promoter sequence (SEQ ID No. 1 ) operably linked to a nucleic acid sequence comprising a Kozak sequence and a nucleic acid sequence encoding a codon-optimized Ebola virus glycoprotein (SEQ ID No. 2).
  • a purified or isolated functional promoter element having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the nucleotide sequence as set forth in SEQ ID No. 1.
  • the promoter is considered to be 'functional' if the promoter has 10%, 20%, 25%, 35% 45%, 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression, preferably 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression when compared to the promoter cassette described herein as SEQ ID No.1 when operably linked to a substantially identical reporter gene, transfected into a substantially similar ceil line and grown under identical conditions, typically, conditions suitable for the expression of the report gene from the promoter, that is, expression driven by the promoter.

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Abstract

A modified CAG promoter which is capable of driving high levels of expression of sequences of interest inserted downstream therefrom is herein described.

Description

OPTIMIZED PROMOTER SEQUENCE PRiOR APPLICATION INFORMATION
The instant application claims the benefit of US Provisional Patent Application 61/086,948, filed August 7, 2008.
BACKGROUND OF THE INVENTION
The Kozak consensus sequence or Kozak sequence plays a major role in the initiation of translation and has the consensus gcc{A/G)ccAUGG.
The CAG promoter comprises a hybrid CMV enhancer coupled to a modified chicken β-actin promoter.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a nucleotide sequence encoding a CAG promoter sequence as set forth in SEQ ID No. 1. According to a second aspect of the invention, there is provided a nucleotide sequence comprising the CAG promoter sequence as set forth in SEQ ID No. 1 operably linked to a nucleic acid sequence comprising a Kozak sequence and a nucleic acid sequence encoding a codon-optimized Ebola virus glycoprotein as set forth in SEQ ID No. 2. According to a third aspect of the invention, there is provided a purified or isolated functional promoter element having at least 70% or more identity with the nucleotide sequence as set forth in SEQ ID No. 1.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1._pCAGaIpha Sequence
Figure 2. Codon Optimized Ebola Glycoprotein Sequence
Figure 3. Bar graphs showing expression intensity of EGFP reporter gene in transfected HEK 293T cells as determined by FACS. Portions of the 5' untranslated region of pCAGGS downstream of the CAG promoter were systematically removed generating pCAGGSΔ764-eGFP, pCAGGSΔ829-eGFP and pCAGGSΔ947-eGFP. Assays were performed in triplicate and repeated twice, the data shown is from one experiment. Error bars represent the standard deviation of the data. * p<0.001.
Figure 4 shows the construction of pCAGGSΔ829. The CAG promoter of the highly efficient expression plasmid pCAGGS is comprised of a CMV-[E enhancer coupled with a modified chicken-beta actin promoter. A minimal promoter sequence was identified following a deletion of 829 base pairs in the intron region using restriction enzyme Eco47ll! and Xbal, we termed this vector pcag-alpha.
Figure 5 shows the construction of pCAGGSΔ764. The CAG promoter of the highly efficient expression plasmid pCAGGS is comprised of a CMV-IE enhancer coupled with a modified chicken-beta actin promoter. We deleted 764 base pairs in the intron region using restriction enzymes PspOMl and Xbai. The cohesive ends were filled in and ligated together using Klenow fragment and T4 DNA ligase.
Figure 6 shows the construction of pCAGGSΔ947. The CAG promoter of the highly efficient expression plasmid pCAGGS is comprised of a CMV-IE enhancer coupled with a modified chicken-beta actin promoter. We deleted 947 base pairs in the intron region using restriction enzymes Eco47III and Acc65l. The cohesive ends were filled in and ligated together using Klenow fragment and T4 DNA ligase
DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
Described herein is an expression cassette comprising an optimized CAG promoter sequence (SEQ ID No. 1 ), shown in Figure 1. The promoter sequence was generated by deleting 829 base pairs using restriction enyzmes Eco47!ll and Xbal, filling the ends with Klenow and then religated the vector carrying the CAG promoter sequence with T4 Ligase, as shown in Figure 4. As can be seen from Figure 3, this deletion resulted in a promoter element having greater efficiency compared to a 'wild type1 construct and other constructs containing a larger deletion. The construction of the 'wild type' element is described in Niwa et al., 1991 , Gene 108:193-200, which is incorporated herein by reference in its entirety, particularly for the details on the construction of the expression vector). The details of the construction of the other plasmids are shown in Figures 5 and 6. Specifically, Figure 5 shows the construction of pCAGGSΔ764 in which 764 base pairs in the intron region were deleted using restriction enzymes PspOM! and Xbal. Figure 6 shows the construction of pCAGGSΔ947 in which 947 base pairs in the intron region were deleted using restriction enzymes Eco47ll! and Acc65l. In both cases, the cohesive ends were filled in and figated together using Klenow fragment and T4 DNA ligase
As will be appreciated by one of skill in the art, such a promoter element can be used to drive high level expression of any gene of interest.
As will be appreciated by one skilled in the art, Figure 3 provides considerable information useful for the construction of similar promoter cassettes. Specifically, one of skill in the art having learned that an approximately 829 base pair deletion produces a promoter having increased expression whereas the 949 base pair deletion negates this increased expression can use this information together with methods known in the art to produce related promoter cassettes without undue experimentation. For example, one of skill in the art could easily construct a series of nested deletions using any of a variety of means known in the art such as for example methods allowing progressive deletion such as nuclease-based methods, restriction enzyme digests, sub-cloning and the like to delineate the exact location of the one or more 'negative' element(s) contained within the 829 base pair deletion fragment responsible for repression of transcriptional expression as well as the location of the one or more 'positive' element(s) located between the end-points of the 829 base pair deletion fragment and the 949 base pair deletion fragment. Furthermore, one of skill in the art could also easily envision other constructs for example constructs containing a deletion that wouid preserve the 'phasing' or relative orientation between the upstream promoter and the transcriptional start site and/or which contained slightly larger or somewhat smaller deletions but which still retained the promoter activity described herein. It is noted that the determination of the location of the positive element could of course be easily carried out using the construct described herein. It is accordingly held that constructs such as those described above which are functionally similar, function (that is, produce a similar but not necessarily identical level of expression, for example, a promoter cassette having 10%, 20%, 25%, 35% 45%, 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression, preferably 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression when compared to the promoter cassette described herein, wherein both constructs are transformed or transfected into the same or a similar cell line and expression is detected under similar conditions) to applicant's construct.
Also described is a nucleotide sequence comprising a Kozak sequence and a nucleotide sequence encoding an Ebola virus glycoprotein. Shown in Figure 2 is such a sequence wherein the codons encoding the Ebola virus glycoprotein have been selected so as to optimize translational efficiency of the Ebola virus glycoprotein in for example HEK 293T cells (SEQ ID No. 2).
In a preferred embodiment, the expression cassette comprises a nucleotide sequence encoding the CAG promoter sequence (SEQ ID No. 1 ) operably linked to a nucleic acid sequence comprising a Kozak sequence and a nucleic acid sequence encoding a codon-optimized Ebola virus glycoprotein (SEQ ID No. 2).
In a further embodiment of the invention, there is provided a purified or isolated functional promoter element having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the nucleotide sequence as set forth in SEQ ID No. 1. It is of note that the promoter is considered to be 'functional' if the promoter has 10%, 20%, 25%, 35% 45%, 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression, preferably 50%, 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more expression when compared to the promoter cassette described herein as SEQ ID No.1 when operably linked to a substantially identical reporter gene, transfected into a substantially similar ceil line and grown under identical conditions, typically, conditions suitable for the expression of the report gene from the promoter, that is, expression driven by the promoter.
While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

Claims

1. A purified or isolated functional promoter element having at least 70% identity with the nucleotide sequence as set forth in SEQ ID No. 1.
2. The promoter element according to claim 1 having at least 80% identity with the nucleotide sequence as set forth in SEQ ID No. 1.
3. The promoter element according to claim 1 having at least 85% identity with the nucleotide sequence as set forth in SEQ ID No. 1.
4. The promoter element according to claim 1 having at least 90% identity with the nucleotide sequence as set forth in SEQ ID No. 1.
5. The promoter element according to claim 1 having at least 95% identity with the nucleotide sequence as set forth in SEQ ID No. 1.
6. The promoter element according to claim 1 having at least 99% identity with the nucleotide sequence as set forth in SEQ ID No. 1.
7. A nucleotide sequence encoding a CAG promoter sequence as set forth in SEQ ID No. 1.
8. A nucleotide sequence comprising the CAG promoter sequence as set forth in SEQ ID No. 1 operably linked to a nucleic acid sequence comprising a Kozak sequence and a nucleic acid sequence encoding a codon-optimized Ebola virus glycoprotein as set forth in SEQ ID No. 2.
PCT/CA2009/001094 2008-08-07 2009-08-07 Optimized promoter sequence WO2010015079A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US13/057,529 US8663981B2 (en) 2008-08-07 2009-08-07 Optimized promoter sequence
JP2011521414A JP5670330B2 (en) 2008-08-07 2009-08-07 Optimized promoter sequence
NZ591439A NZ591439A (en) 2008-08-07 2009-08-07 pCAGGS VECTOR COMPRISING AN 829 BASE PAIR DELETION IN THE CHICKEN-BETA ACTIN INTRON WHICH RESULTS IN ENHANCED EXPRESSION OF A GENE OF INTEREST
EP09804421.7A EP2318537B1 (en) 2008-08-07 2009-08-07 Optimized promoter sequence
AU2009279334A AU2009279334B2 (en) 2008-08-07 2009-08-07 Optimized promoter sequence
CA2733104A CA2733104C (en) 2008-08-07 2009-08-07 Optimized promoter sequence

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US8694808P 2008-08-07 2008-08-07
US61/086,948 2008-08-07

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CA3100070A1 (en) * 2018-05-18 2019-11-21 Universite Laval Vectors for dna vaccination
MX2021002041A (en) * 2018-08-30 2021-07-21 Res Inst Nationwide Childrens Hospital Gene therapy for the treatment of galactosemia.
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WO2015033086A1 (en) * 2013-09-04 2015-03-12 Fujifilm Diosynth Biotechnologies Uk Limited Dna construct
WO2019094061A1 (en) * 2017-11-07 2019-05-16 The University Of North Carolina At Chapel Hill Optimized aga genes and expression cassettes and their use
US11491241B2 (en) 2017-11-07 2022-11-08 The University Of North Carolina At Chapel Hill Optimized AGA genes and expression cassettes and their use

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