GENE THERAPY FOR NEURODEGENERATIVE DISORDERS USING POLYNUCLEOTIDE SILENCING AND REPLACEMENT

INCORPORATION OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, named APRES1120_1WO_Sequence_Listing.txt, was created on Jan. 29, 2021, and is 194,755 bytes in size. The file can be accessed using Microsoft Word on a computer that uses Windows OS.

TECHNICAL FIELD

The present disclosure relates generally to gene therapy for neurodegenerative disorders, and more specifically to expression cassettes and polynucleotides for delivery of therapeutic agents.

BACKGROUND

Alzheimer's disease (AD), also referred to as Alzheimer's, is a chronic neurodegenerative disease that is the cause of a majority of neurodegenerative dementia. Symptoms include difficulty with memory, problems with language, disorientation, mood swings, loss of motivation, and other behavioral problems such as withdrawal from family and society. Bodily functions are gradually lost, ultimately leading to death. Although the disease can last for more than ten years, the average life expectancy is three to nine years following diagnosis. Familial AD (FAD) characterizes families that have more than one member with AD and usually implies multiple affected persons in more than one generation. Early-onset FAD (EOFAD) refers to families in which onset is consistently before age 60 to 65 years and often before age 55 years.

AD appears pathologically as extracellular amyloid plaques and intracellular neurofibrillary tangles in the brain. Although the cause for most cases of AD is not known, genetic factors contribute to development of disease. Early onset familial AD is characterized by autosomal dominant inheritance and by disease onset before age 65.

There is a need for compositions and methods for the treatment of neurodegenerative diseases such as AD, including effective gene and combination therapies.

SUMMARY

The present disclosure relates to polynucleotides, expression cassettes and vectors comprising such polynucleotides and/or expression cassettes for the treatment of neurodegenerative disorders. More specifically, the polynucleotides, expression cassettes and vectors utilized in the present disclosure comprise a) a first polynucleotide sequence that encodes one or more short hairpin RNAs (shRNAs) or micro interfering RNAs (miRNAs) having sufficient sequence complementarity with mRNA expressed from an endogenous presenilin 1 (PSEN1) or presenilin 2 (PSEN2) gene, to hybridize to that mRNA and inhibit expression of the encoded presenilin 1 (PSEN1) or presenilin 2 (PSEN2) protein, or the combination thereof, and b) a second polynucleotide sequence encoding a wild-type PSEN1 or PSEN2 protein, or a combination thereof. The wild-type PSEN1 or PSEN2 encoded by the second polynucleotide is expressed utilizing control sequences that are present in the expression cassette and/or vector harboring them, as opposed to endogenous control sequences. The mRNA expressed from the second polynucleotide sequence must be resistant to suppression by the short hairpin RNAs (shRNAs) or micro interfering RNAs (miRNAs) encoded by the first polynucleotide sequence. Therefore, the simultaneous expression of the wild-type PSEN1 or PSEN2 protein results in the replacement of the endogenously expressed PSEN1 or PSEN2 protein.

Presenilins can harbor mutations which cause autosomal-dominant gain of toxic function. Such mutations are distributed throughout the coding sequence for both PSEN1 and its homolog PSEN2. The ability to simultaneously suppress autosomal-dominant mutated presenilins and express the wild-type gene eliminates the need to specifically target the mutant allele. Therefore, the polynucleotides, expression cassettes, and vectors of the disclosure, and the compositions and methods of the disclosure employing them are useful for halting and/or ameliorating damage associated with mutant PSEN1 or PSEN2 or the combination thereof.

The ability of the replacement wild-type PSEN1 or PSEN2 to avoid being targeted and suppressed by the one or more shRNAs or miRNAs will depend on which locations PSEN1 or PSEN2 mRNA sequences are targeted by the shRNAs or miRNAs and what codons are used in the replacement wild-type PSEN1 or PSEN2 coding sequence. If all of the designed shRNAs or miRNAs target non-coding regions of endogenous PSEN1 or PSEN2 mRNA, then the replacement PSEN1 or PSEN2 polynucleotide sequence can be any sequence that encodes wild-type PSEN1 or PSEN2 including, but not limited to, the endogenous human PSEN1 or PSEN2 coding sequence, or a sequence wherein some or all of the codons are altered based on the redundancy of the genetic code in order to increase expression, e.g., a fully or partially codon-optimized, wild-type PSEN1 or PSEN2 polynucleotide sequence. If some or all of the shRNAs or miRNAs target coding regions of endogenous PSEN1 or PSEN2 mRNA, then the corresponding coding regions in the replacement PSEN1 or PSEN2 polynucleotide sequence must be modified so the mRNA expressed is not targeted by any of the shRNAs or miRNAs. This is achieved by modifying endogenous codons using the redundancy of the genetic code to reduce homology/complementarity of the mRNA expressed with the shRNA or miRNA sequences.

In some embodiments, the polynucleotides, expression cassettes, vectors, compositions and methods disclosed herein are useful for suppressing an endogenous PSEN1 protein while at the same time increasing levels of a wild-type PSEN1 protein. Suppression of endogenous PSEN1 protein typically is achieved through the use of one or more antisense oligonucleotides that binds to a mRNA expressed from the endogenous PSEN1 gene thereby decreasing the level of such mRNA and/or inhibiting its translation into protein. In some aspects of these embodiments, the antisense oligonucleotide is an antisense RNA encoded by a DNA sequence that is administered to a subject as part of an expression cassette or vector. Such antisense RNA include shRNAs, miRNAs, or single-stranded antisense RNAs. In alternate aspects of these embodiments, the antisense oligonucleotide is delivered directly to the subject. Such antisense oligonucleotides include siRNAs, antisense DNA oligonucleotides, external guide sequence oligonucleotides and alternate splicer oligonucleotides. In some aspects of these embodiments, a non-toxic dual function vector is provided that is capable of expressing both an antisense RNA and a wild-type PSEN1 whose expression is not suppressed that antisense RNA. In some embodiments, the antisense oligonucleotide is administered concurrently with a vector encoding wild-type PSEN1, the expression of which is not suppressed by the antisense oligonucleotide provided. In still other aspects of these embodiments, a first vector comprising a DNA sequence encoding an antisense RNA is administered concurrently with a second vector comprising a DNA sequence encoding wild-type PSEN1, the expression of which is not suppressed by the antisense RNA.

In some embodiments, the polynucleotides, expression cassettes, vectors, compositions and methods disclosed herein are useful for suppressing an endogenous PSEN2 protein while at the same time increasing levels of a wild-type PSEN2. Suppression of endogenous PSEN2 protein typically is achieved through the use of one or more antisense oligonucleotides that binds to a mRNA expressed from the endogenous PSEN2 gene thereby decreasing the level of such mRNA and/or inhibiting its translation into protein. In some aspects of these embodiments, the antisense oligonucleotide is an antisense RNA encoded by a DNA sequence that is administered to a subject as part of an expression cassette or vector. Such antisense RNA include shRNAs, miRNAs, or single-stranded antisense RNAs. In alternate aspects of these embodiments, the antisense oligonucleotide is delivered directly to the subject. Such antisense oligonucleotides include siRNAs, antisense DNA oligonucleotides, external guide sequence oligonucleotides and alternate splicer oligonucleotides. In some aspects of these embodiments, a non-toxic dual function vector is provided that is capable of expressing both an antisense RNA and a wild-type PSEN2 whose expression is not suppressed that antisense RNA. In some embodiments, the antisense oligonucleotide is administered concurrently with a vector encoding wild-type PSEN2, the expression of which is not suppressed by the antisense oligonucleotide provided. In still other aspects of these embodiments, a first vector comprising a DNA sequence encoding an antisense RNA is administered concurrently with a second vector comprising a DNA sequence encoding wild-type PSEN2, the expression of which is not suppressed by the antisense RNA.

In some embodiments, an expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently targets a coding region or a non-coding region of an endogenous mRNA expressed from each of a human wild-type and a mutant presenilin 1 (PSEN1) or each of a human wild-type and a mutant presenilin 2 (PSEN2), wherein each of the polynucleotide sequences encoding the one or more shRNA or miRNA is operably linked to one or more first promoters; and (II) a second polynucleotide encoding a wild-type PSEN1 or PSEN2 amino acid sequence, wherein the mRNA expressed from the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first polynucleotide; and wherein the second polynucleotide is operably linked to a second promoter. The first polynucleotide may be positioned anywhere in the expression cassette relative to the PSEN1 or PSEN2 coding sequence, as long as its location does not prevent expression of the PSEN1 or PSEN2 coding sequence (i.e., 5′ to the coding sequence, 3′ to the coding sequence, or within an intron that could exist in the second promoter.

In certain embodiments, an expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently comprises one of: a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, or nucleotides 448-529 of SEQ ID NO:71; b) a modified version of any of the foregoing SEQ ID NOs, wherein the modification is 1, 2, 3, or 4 nucleotide changes; or c) a 19-21 base nucleotide sequence comprising 7 or more consecutive bases taken from the 5′ or 3′ end of any of the foregoing SEQ ID NOs or the modified version thereof, wherein the 19-21 base nucleotide sequence comprises no more than 4 mismatches with a corresponding portion of an endogenous PSEN1 mRNA, wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (II) a second polynucleotide encoding a wild-type presenilin 1 (PSEN1) protein, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first polynucleotide, and wherein the second polynucleotide is operably linked to a second promoter.

SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO:35, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, and nucleotides 448-529 of SEQ ID NO:71, each encode RNA that target sequences in the non-coding portion of PSEN1 mRNA. SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, and SEQ ID NO:47 each encode RNA that target sequences in the coding portion of PSEN1 mRNA. SEQ ID NO:33, SEQ ID NO:35, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, and nucleotides 448-529 of SEQ ID NO:71 each encode a miRNA. SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, and SEQ ID NO:47 each encode a shRNA.

Each of the 1, 2, 3, or 4 nucleotide changes in the modified version of any of the above SEQ ID NOs is independently a nucleotide substitution, deletion, or addition and results in a mismatch with endogenous wild-type PSEN1 mRNA. The additional nucleotides required for a 19-21 base nucleotide sequence comprising 7 or more consecutive bases taken from the 5′ or 3′ end of any of the foregoing SEQ ID NOs or the modified version thereof are those that are capable of hybridizing to the region of PSEN1 mRNA immediately 5′ or 3′, respectively, to the regions of PSEN1 mRNA to which the 7 or more consecutive bases bind, while still allowing for up to 4 mismatches in the entire 19-21 base nucleotide sequence. For example, SEQ ID NO:1 hybridizes to nucleotides 94-115 of PSEN1 mRNA (using numbering in NM_000021.4) (See Table 2, herein). Thus, an example of a 19-21 base nucleotide sequence taken from the 5′ end of SEQ ID NO:1 would contain nucleotides 2-8 with a perfect complementarity to PSEN1 mRNA and other bases would contain 1, 2, 3, or 4 nucleotide changes.

In some embodiments, and expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently comprises one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, or nucleotides 448-529 of SEQ ID NO:71, wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (II) a second polynucleotide encoding a wild-type presenilin 1 (PSEN1) protein, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first polynucleotide, and wherein the second polynucleotide is operably linked to a second promoter.

In some embodiments, an expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently comprises one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO: 35,SEQ ID NO:42, SEQ ID NO:43 SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, or nucleotides 448-529 of SEQ ID NO:71, and wherein at least one shRNA or miRNAs comprises one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 42, or SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, nucleotides 497-517 of SEQ ID NO:68, nucleotides 497-517 of SEQ ID NO:69, nucleotides 497-517 of SEQ ID NO:70, nucleotides 497-517 of SEQ ID NO:71, wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (ii) a second polynucleotide encoding a wild-type presenilin 1 (PSEN1) protein, wherein the second polynucleotide expresses a mRNA that encodes a human, wild-type PSEN1 and is not targeted by any of the shRNAs or miRNAs, and wherein the second polynucleotide is operably linked to a second promoter. In some aspects of these embodiments, the second polynucleotide expresses a mRNA encoding wild-type PSEN1 that is codon modified as compared to the coding portion of the endogenous, human, wild-type PSEN1 mRNA. In more specific aspects of these embodiments, the second polynucleotide expresses a mRNA that comprises a sufficient number of modified codons in those coding regions that are targeted by the shRNAs or miRNAs to prevent such shRNAs or miRNAs from targeting the mRNA expressed from the second polynucleotide. Typically, modifying more than 4 nucleotides in the mRNA coding sequence of the second polynucleotide to reduce homology/complementarity with the shRNA or miRNAs will prevent targeting. In even more specific aspects of these embodiments, the second polynucleotide sequence is SEQ ID NO:41.

In certain embodiments, an expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently comprises one of: a) SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78; b) a modified version of any of the foregoing SEQ ID NOs, wherein the modification is 1, 2, 3, or 4 nucleotide changes; or c) a 19-21 base nucleotide sequence comprising 7 or more consecutive bases taken from the 5′ or 3′ end of any of the foregoing SEQ ID NOs or the modified version thereof, wherein the 19-21 base nucleotide sequence comprises no more than 4 mismatches with a corresponding portion of an endogenous PSEN1 mRNA, wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (II) a second polynucleotide encoding a wild-type presenilin 2 (PSEN2) protein, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first polynucleotide, and wherein the second polynucleotide is operably linked to a second promoter.

SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO:36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78 each encode RNA that target sequences in the non-coding portion of PSEN2 mRNA. SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 each encode RNA that target sequences in the coding portion of PSEN2 mRNA. SEQ ID NO:34, SEQ ID NO:36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78 represent miRNA coding sequences. Each of the 1, 2, 3, or 4 nucleotide changes in the modified version of any of the above SEQ ID NOs is independently a nucleotide substitution, deletion, or addition and results in a mismatch with endogenous wild-type PSEN2 mRNA. The additional nucleotides required for a 19-21 base nucleotide sequence comprising 7 or more consecutive bases taken from the 5′ or 3′ end of any of the foregoing SEQ ID NOs or the modified version thereof are those that are capable of hybridizing to the region of PSEN2 mRNA immediately 5′ or 3′, respectively, to the regions of PSEN2 mRNA to which the 7 or more consecutive bases bind, while still allowing for up to 4 mismatches in the entire 19-21 base nucleotide sequence. For example, SEQ ID NO:20 hybridizes to nucleotides 110-135 of PSEN2 mRNA (using numbering in NM 000447.3) (See Table 3, herein). Thus, an example of a 19-21 base nucleotide sequence taken from the 5′ end of SEQ ID NO:20 would contain nucleotides 2-8 with a perfect complementarity to PSEN2 mRNA and other bases would contain 1, 2, 3 or 4 nucleotide changes. Similarly, an example of a 19-21 base nucleotide sequence taken from the 3′ end of SEQ ID NO:21 would contain nucleotides 2-8 with a perfect complementarity to PSEN2 mRNA and other bases would contain 1, 2, 3, or 4 nucleotide changes.

In certain embodiments, an expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently comprises one of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78, wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (II) a second polynucleotide encoding a wild-type presenilin 2 (PSEN2) protein, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first polynucleotide, and wherein the second polynucleotide is operably linked to a second promoter. SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO:36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78 each encode RNA that target sequences in the non-coding portion of PSEN2 mRNA. SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 each encode RNA that target sequences in the coding portion of PSEN2 mRNA. SEQ ID NO:34, SEQ ID NO:36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78 represent miRNA coding sequences.

In some embodiments, an expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently comprises one of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78, wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (ii) a second polynucleotide encoding a wild-type presenilin 2 (PSEN2) protein, wherein the second polynucleotide expresses any mRNA that encodes a human, wild-type PSEN2, and wherein the second polynucleotide is operably linked to a second promoter. In some aspects of these embodiments, the second polynucleotide expresses a mRNA, wherein the coding portion of the mRNA has the same polynucleotide sequence as endogenous, human, wild-type PSEN2 mRNA. In other aspects of these embodiments, the second polynucleotide expresses a mRNA encoding wild-type PSEN2, wherein the coding portion of the mRNA has a polynucleotide sequence wherein one or more codons have been modified or optimized as compared to the coding portion of the endogenous, human, wild-type PSEN2 mRNA. In more specific aspects of these embodiments, the second polynucleotide sequence is SEQ ID NO:40.

In some embodiments, an expression cassette comprises: (I) a first polynucleotide encoding one or more shRNAs or miRNAs, each of which independently comprises one of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, and nucleotides 448-529 of SEQ ID NO:78; and wherein at least one shRNA or miRNAs comprises one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, nucleotides 497-517 of SEQ ID NO:76, nucleotides 497-517 of SEQ ID NO:77, or nucleotides 497-517 of SEQ ID NO:78, wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (ii) a second polynucleotide encoding a wild-type presenilin 2 (PSEN2) protein, wherein the second polynucleotide expresses a mRNA that encodes a human, wild-type PSEN2 and is not targeted by any of the shRNAs or miRNAs, and wherein the second polynucleotide is operably linked to a second promoter. In some aspects of these embodiments, the second polynucleotide expresses a mRNA encoding wild-type PSEN2 that is codon modified as compared to the coding portion of the endogenous, human, wild-type PSEN2 mRNA. In more specific aspects of these embodiments, the second polynucleotide expresses a mRNA that comprises a sufficient number of modified codons in those coding regions that are targeted by the shRNAs or miRNAs to prevent such shRNAs or miRNAs from targeting the mRNA expressed from the second polynucleotide.

The one or more first promoters drive expression of each shRNA or miRNA encoding sequence. Each shRNA or miRNA encoding sequence may be driven by the same or a different first promoter. When driven by the same first promoter, expression of two or more shRNA or miRNA encoding sequences may be driven by separate copies of the same first promoter or by a single copy of that first promoter. When driven by a single copy of a first promoter, two or more shRNA or miRNA encoding sequences will be located in tandem to one another in the expression cassette such that a single first promoter can drive expression of each one of those shRNA or miRNA encoding sequences. Similarly, the second promoter, which drives expression of the replacement, wild-type PSEN1 or PSEN2, may also drives expression of the shRNA or miRNA encoding sequences (i.e., the first promoter and the second promoter are the same). When driven by a single promoter, the shRNA or miRNA encoding sequences will be located in tandem with the PSEN1 or PSEN2 coding sequence in the expression cassette such that such single first promoter can drive expression of both the shRNA or miRNA encoding sequence and the PSEN1 or PSEN2 coding sequence. In certain aspects, a single promoter can drive expression of two or more shRNA or miRNA and PSEN1 or PSEN2. In some embodiments, at least one of the one or more first promoters or second promoters is a RNA polymerase III promoter or a RNA polymerase II promoter. In some more specific aspects of these embodiments, the RNA polymerase III promoter is selected from U6 promoter, a U61 promoter, a U69 promoter, a H1 promoter, or any combination thereof. In some aspects of these embodiments, at least one of the one or more first promoters or second promoter is a RNA polymerase II promoter that is a neuron-specific promoter. In some more specific aspects of these embodiments, the second promoter is a RNA polymerase II promoter that is a neuron-specific promoter. In other more specific aspects of these embodiments, the second promoter is a RNA polymerase II promoter that is a ubiquitous promoter.

In some embodiments, the disclosure provides a vector comprising any of the expression cassettes disclosed herein.

In some embodiments, the disclosure provides a set of vectors, comprising (a) a first vector comprising an expression cassette comprising a first polynucleotide encoding one or more shRNAs or miRNAs targeting either a coding region or a non-coding region of a mRNA translated from each of a human wild-type and a mutant presenilin 1 (PSEN1), or from each of a human wild-type and a mutant presenilin 2 (PSEN2), wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (b) a second vector comprising a second polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence or a wild-type presenilin 2 (PSEN2) amino acid sequence, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first vector; and wherein the second polynucleotide is operably linked to a second promoter.

In some embodiments, the disclosure provides a set of vectors, comprising (a) a first vector comprising an expression cassette comprising (a) a first polynucleotide encoding one or more shRNAs or miRNAs targeting either a coding region or a non-coding region of a mRNA translated from each of a human wild-type and a mutant presenilin 1 (PSEN1), wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (b) a second polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first polynucleotide; and wherein the second polynucleotide is operably linked to a second promoter.

In some embodiments, the disclosure provides a set of vectors, comprising (a) a first vector comprising an expression cassette comprising (a) a first polynucleotide encoding one or more shRNAs or miRNAs targeting either a coding region or a non-coding region of a mRNA translated from each of a human wild-type and a mutant presenilin 2 (PSEN2), wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and (b) a second polynucleotide encoding a wild-type presenilin 2 (PSEN2) amino acid sequence, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first polynucleotide; and wherein the second polynucleotide is operably linked to a second promoter.

In some embodiments of a set of vectors, each the encoded shRNAs or miRNAs in the first vector independently comprises one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, or nucleotides 448-529 of SEQ ID NO:71. In some aspects of these embodiments, each of the encoded shRNAs or miRNAs each of which independently comprises one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO: 35, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, or nucleotides 448-529 of SEQ ID NO:71. In other aspects of these embodiments, each of the encoded shRNAs or miRNAs each of which independently comprises one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO: 35,SEQ ID NO:42, SEQ ID NO:43 SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, or nucleotides 448-529 of SEQ ID NO:71; and wherein at least one shRNA or miRNAs comprises one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 42, or SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, nucleotides 497-517 of SEQ ID NO:68, nucleotides 497-517 of SEQ ID NO:69, nucleotides 497-517 of SEQ ID NO:70, or nucleotides 497-517 of SEQ ID NO:71.

In some embodiments of a set of vectors, when each of the shRNAs or miRNAs in the first vector targets a non-coding region present in an endogenous PSEN1 mRNA, the second polynucleotide in the second vector expresses a mRNA, wherein the coding portion of the mRNA has the same polynucleotide sequence as endogenous, human, wild-type PSEN1 mRNA. In other aspects of these embodiments, the second polynucleotide expresses a mRNA, wherein the coding portion of the mRNA has a polynucleotide sequence wherein one or more codons have been modified or optimized as compared to the coding portion of the endogenous, human, wild-type PSEN1 mRNA. In more specific aspects of these embodiments, the second polynucleotide sequence is SEQ ID NO:39. In other more specific aspects of these embodiments, the second polynucleotide sequence is SEQ ID NO:48.

In some embodiments of a set of vectors, when at least one of the shRNAs or miRNAs in the first vector targets a coding region present in an endogenous PSEN1 mRNA, the second polynucleotide in the second vector expresses a mRNA that is codon modified as compared to the coding portion of the endogenous, human, wild-type PSEN1 mRNA. In more specific aspects of these embodiments, the second polynucleotide expresses a mRNA that comprises a sufficient number of modified codons in those coding regions that are targeted by the shRNAs or miRNAs to prevent such shRNAs or miRNAs from targeting the mRNA expressed from the second polynucleotide. Typically, modifying a sufficient number of codons to create more than 4 mismatched nucleotides with the shRNA or miRNA will prevent targeting. In even more specific aspects of these embodiments, the second polynucleotide sequence is SEQ ID NO:41.

In some embodiments of a set of vectors, each the encoded shRNAs or miRNAs in the first vector independently comprises one of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, or nucleotides 448-529 of SEQ ID NO:78. In some aspects of these embodiments, each the encoded shRNAs or miRNAs each of which independently comprises one of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, or nucleotides 448-529 of SEQ ID NO:78. In other aspects of these embodiments, each the encoded shRNAs or miRNAs each of which independently comprises one of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, or nucleotides 448-529 of SEQ ID NO:78; and wherein at least one shRNA or miRNAs comprises one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, nucleotides 497-517 of SEQ ID NO:76, nucleotides 497-517 of SEQ ID NO:77, or nucleotides 497-517 of SEQ ID NO:78.

In some embodiments of a set of vectors, when each of the shRNAs or miRNAs in the first vector targets a non-coding region present in an endogenous PSEN2 mRNA, the second polynucleotide in the second vector expresses a mRNA, wherein the coding portion of the mRNA has the same polynucleotide sequence as endogenous, human, wild-type PSEN2 mRNA. In other aspects of these embodiments, the second polynucleotide expresses a mRNA, wherein the coding portion of the mRNA has a polynucleotide sequence wherein one or more codons have been modified or optimized as compared to the coding portion of the endogenous, human, wild-type PSEN2 mRNA. In more specific aspects of these embodiments, the second polynucleotide sequence is SEQ ID NO:40.

In some embodiments of a set of vectors, when at least one of the shRNAs or miRNAs in the first vector targets a coding region present in an endogenous PSEN2 mRNA, the second polynucleotide in the second vector expresses a mRNA that is codon modified as compared to the coding portion of the endogenous, human, wild-type PSEN2 mRNA. In more specific aspects of these embodiments, the second polynucleotide expresses a mRNA that comprises a sufficient number of modified codons in those coding regions that are targeted by the shRNAs or miRNAs to prevent such shRNAs or miRNAs from targeting the mRNA expressed from the second polynucleotide. Typically, modifying a sufficient number of codons to create more than 4 mismatched nucleotides with the shRNA or miRNA will prevent targeting.

Each vectors in any of the foregoing embodiments can be a viral vector, such as an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector. An AAV vector can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVDJ, AAVrh10, AAV11, AAV12, AAV2/1, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/11, or AAV2/12, and capsid engineered adeno-associated viruses with hybrid capsids merging portions of two or more natural AAVs and/or point mutations of natural AAVs to modify tropism or evade immune detection such as PHP.B, and PHP.B derivatives [PHP.eR, PHP.S], AAV8[K137R], AAV-TT, rAAV-retro, AAV9.HR, AAV1 CAM mutants, AAV9[586-590] swap mutants. Vectors or sets of vectors can be plasmid vectors with or without carrier such as polyamine.

In other embodiments, provided herein are kits comprising a vector or sets of vectors provided herein.

In other embodiments, an isolated polynucleotide of SEQ ID NO:41 is provided.

In still other embodiments is provided a kit comprising: (a) one or more antisense oligonucleotides, wherein each antisense oligonucleotide independently targets either a coding region or a non-coding region of an mRNA translated from each of a human wild-type and mutant presenilin 1 (PSEN1), each of a human wild-type or mutant presenilin 2 (PSEN2); and (b) a vector comprising a polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence or a wild-type presenilin 2 (PSEN2) amino acid sequence, wherein the second polynucleotide is not targeted by any of the one or more antisense oligonucleotides; and wherein the polynucleotide is operably linked to a promoter in the vector. In some aspects of these embodiments, each of the one or more antisense oligonucleotides targets either a coding region or a non-coding region of an mRNA translated from each of a human wild-type and mutant presenilin 1 (PSEN1); and the vector comprises a polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence.

In some embodiments of the kit described in the previous paragraph, each of the one or more antisense oligonucleotides is independently selected from a short hairpin RNA (shRNA), a short interfering RNA (siRNA), a micro interfering RNA (miRNA), a small temporal RNA (stRNA) or an endoribonuclease-prepared siRNA (esiRNA). In some aspects of these embodiments, at least one of the one or more antisense oligonucleotides comprises one or more modified nucleobases. In some more specific aspects of these embodiments, each of the one or more modified nucleobases is independently selected from a non-naturally occurring nucleobase, a locked nucleic acids (LNA), or a peptide nucleic acids (PNA).

Yet another embodiment provides methods of treating a neurodegenerative disease, disorder, or condition, wherein the method comprises the step of administering to a subject in need thereof:

(a) either:

- (i) one or more antisense oligonucleotides, wherein each antisense oligonucleotide independently targets either a coding region or a non-coding region of an mRNA translated from each of a human wild-type and mutant presenilin 1 (PSEN1), each of a human wild-type or mutant presenilin 2 (PSEN2), or
- (ii) a vector comprising a first polynucleotide encoding one or more shRNAs or miRNAs targeting either a coding region or a non-coding region of a mRNA translated from each of a human wild-type and a mutant presenilin 1 (PSEN1), or from each of a human wild-type and a mutant presenilin 2 (PSEN2), wherein each of the one or more shRNAs or miRNAs is operably linked to one or more first promoters; and
- (b) a vector comprising a second polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence or a wild-type presenilin 2 (PSEN2) amino acid sequence, wherein the second polynucleotide is not targeted by any of the shRNAs or miRNAs encoded by the first vector; and wherein the second polynucleotide is operably linked to a second promoter.

In some aspects of these embodiments, the first polynucleotide encoding one or more shRNAs or miRNAs and the second polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence or a wild-type presenilin 2 (PSEN2) amino acid sequence are present in the same vector. Such vectors are described above as containing any of the expression vectors disclosed herein. In alternate aspects of these embodiments, the first polynucleotide encoding one or more shRNAs or miRNAs and the second polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence or a wild-type presenilin 2 (PSEN2) amino acid sequence are present in different vectors (i.e., a set of vectors). Such sets of vectors are also disclosed herein. In still other alternate aspects of these embodiments, the targeting an mRNA translated from each of a human wild-type and mutant presenilin 1 (PSEN1), each of a human wild-type or mutant presenilin 2 (PSEN2) is achieved by administering an antisense RNA molecule. Such antisense RNA molecules are also disclosed herein. In certain aspects of these embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease, frontotemporal dementia, frontotemporal lobar degeneration, Pick's disease, Lewy body dementia, memory loss, cognitive impairment, or mild cognitive impairment.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is plasmid map of pAT049.

FIG. 2 is plasmid map of pAT050.

FIG. 3 is plasmid map of pAT051.

FIG. 4 is plasmid map of pAT052.

FIG. 5 is plasmid map of pAT053.

FIG. 6 is plasmid map of pAT054.

FIG. 7 is plasmid map of pAT055.

FIG. 8 is plasmid map of pAT056.

FIG. 9 is plasmid map of pAT057.

FIG. 10 is plasmid map of pAT058.

FIG. 11 is plasmid map of pAT059.

FIG. 12 is plasmid map of pAT060.

FIG. 13 is plasmid map of pAT061.

FIG. 14 is plasmid map of pAT062.

FIG. 15 is a bar graph representing the levels of endogenous PSEN1 (white bars) and plasmid-encoded (exogenous) PSEN1 transcripts (black bars) in HEK293 cells after transfection with different plasmids encoding exogenous PSEN1 and a miRNA targeting sequence that specifically hybridizes to endogenous PSEN1. Exogenous and endogenous transcript levels are compared to untrasnsfected cells, treatment with an empty vector (EV), and treatment with a vector encoding exogenous PSEN1 without any miRNA as controls.

FIGS. 16A and 16B are bar graphs representing the levels of endogenous PSEN1 (FIG. 16A) and plasmid-encoded (exogenous) PSEN2 (FIG. 16B) transcripts in HEK293 cells after transfection with different plasmids encoding exogenous PSEN2 and a miRNA targeting sequence that specifically hybridizes to endogenous PSEN2. Exogenous and endogenous transcript levels are compared to untrasnsfected cells, and treatment with an empty vector (EV).

Definitions

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 pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, recitation of “a cell”, for example, includes a plurality of the cells of the same type. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/−20%, +/−10%, +/−5%, +/−1%, or +/−0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

As used herein, the term “antisense oligonucleotide” means an RNA or a single or double-stranded DNA molecule at least part of which binds to another RNA or DNA (target RNA, DNA) through hybridization. The portion of an antisense oligonucleotide that hybridizes to its target is term the “antisense portion”. For example, if the antisense oligonucleotide is an RNA oligonucleotide, the antisense portion thereof binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA. The antisense oligonucleotides used herein downregulate expression of PSEN1 or PSEN2. The term “antisense oligonucleotide” is meant to include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro interfering RNA (miRNA), siRNA, short hairpin RNA (shRNA), external guide sequence (EGS) oligonucleotides, alternate splicers, and any of the foregoing that comprise one or more modified nucleobases. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

An antisense oligonucleotide is “specifically hybridizable” when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

“Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nucleobase at a certain position of an antisense oligonucleotide is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligomeric oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.

It is understood in the art that the sequence of an antisense oligonucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). The antisense oligonucleotides of the present invention typically contain no more than 4, no more than 3, no more than 2, no more than 1, or no mismatches with the portion of the PSEN1 or PSEN2 nucleic acid sequence to which they are targeted.

The term “mismatch” as used herein refers to: 1) the inability of a nucleotide in an antisense portion of an antisense oligonucleotide to base pair with its target mRNA or vice versa; or 2) the inability of a nucleotide in an antisense portion of an antisense oligonucleotide to base pair with its sense portion in that antisense oligonucleotide. The antisense portion of an antisense oligonucleotide may have a mismatch with its target mRNA or sense portion due to a substitution, deletion or addition of a nucleotide. Each nucleotide that is substituted, deleted, or added is considered a separate mismatch.

As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements—or, as appropriate, equivalents thereof—and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.

The term “expression” as used herein is defined as the transcription of a mRNA from a DNA sequence driven by a promoter and/or translation of a particular amino acid sequence from a mRNA sequence.

As used herein, the term “expression cassette” refers to a DNA sequence that encodes and is capable of producing one or more desired expression products (RNA or protein). Production of such a desired expression product requires the presence of various expression control sequences operatively linked to the DNA sequence encoding that product. Such control sequences include a promoter, as well as other non-coding nucleotide sequences. An expression cassette may include none, some or all of these expression control sequences. If some or all of these expression control sequences are absent from the expression cassette, they are supplied by a vector into which the expression cassette is inserted.

As used herein, a “subject” means a human. The terms, “patient”, “individual” and “subject” are used interchangeably herein. A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., brain tumors) or one or more complications related to the condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to the condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition, e.g. a neurodegenerative condition, can be a subject suspected of having that condition, diagnosed as having that condition, already treated or being treated for that condition, not treated for that condition, or at risk of developing that condition.

The term “polynucleotide” as used herein means a sequence of 20 or more nucleotides. A polynucleotide may RNA, DNA or a hybrid RNA or DNA molecule; and may be single stranded or double stranded. In certain embodiments, a polynucleotide is a single or double-stranded DNA molecule.

The term “target” and various forms thereof (e.g., “targeted”, “targeting”) with respect to a nucleic acid sequence e.g. a mRNA encoded by PSEN1 or PSEN2, means a portion of that nucleic acid sequence to which an antisense oligonucleotide is designed to specifically hybridize resulting in reduced or eliminated expression of that nucleic acid sequence.

The term “wild-type” with respect to PSEN1 means the amino acid sequence encoded by SEQ ID NO:39, whether present endogenously within the subject or encoded by a polynucleotide administered to the subject. The term “wild-type” with respect to PSEN2 means the amino acid sequence encoded by SEQ ID NO:40, whether present endogenously within the subject or encoded by a polynucleotide administered to the subject.

The term “endogenous” as used herein means a form of a gene, or mRNA that is naturally found in a human subject. An endogenous gene or mRNA encoding PSEN1 or PSEN2 includes sequences encoding wild-type PSEN1 or PSEN2, as well as those encoding mutant forms of PSEN1 or PSEN2 that are naturally found in a human subject.

The term “regulatory element” refers to a non-coding portion of a polynucleotide or vector that is necessary for and/or enhances expression of a coding portion of that polynucleotide. Examples of regulatory elements include, without limitation, promoters, enhancers, polyadenylation signals, chromatin insulators, translation initiation sequences such as strong and weak Kozak signal sequences and internal ribosomal entry sites, mRNA stability sequences, sequences that influence mRNA processing such as splicing and cleavage, sequences that influence mRNA export from the nucleus and/or mRNA retention, posttranslational response elements, non-coding sequences such as introns and untranslated regions (UTRs), poly A sequences, repressors, silencers, terminators, and others.

As used herein, “operably linked,” “operable linkage,” “operatively linked,” or grammatical equivalents thereof refer to juxtaposition of genetic elements, e.g., typically a polynucleotide encoding an expression product, i.e., a protein or RNA, and a non-coding regulatory element, wherein the elements are in a relationship permitting them to operate in the expected manner. For example, a promoter is “operably linked” to a polynucleotide encoding a desired expression product when they are juxtaposed with respect to one another such that promoter can drive expression of the polynucleotide.

The term “codon modified” as used herein means a DNA or RNA sequence encoding the same amino acid sequence as a naturally occurring protein (i.e., wild-type PSEN1 or wild-type PSEN2), wherein, due to the redundancy of the genetic code, at least one codon has been altered as compared to the endogenous DNA or RNA encoding that protein.

The term “codon optimized” as used herein means a codon modified DNA or RNA sequence, wherein the modified codons are selected from the preferred codons or most preferred codons set forth in Table 1.

Where any amino acid sequence is specifically referred to by a Swiss Prot. or GENBANK Accession number, the sequence is incorporated herein by reference. Information associated with the accession number, such as identification of signal peptide, extracellular domain, transmembrane domain, promoter sequence and translation start, is also incorporated herein in its entirety by reference.

Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to human homologs or mutated forms for which the compositions and methods disclosed herein are applicable.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

DETAILED DESCRIPTION

The present disclosure provides compositions that comprise (1) antisense oligonucleotides (or polynucleotides that encode them) for silencing endogenous forms of PSEN1 and/or PSEN2 mRNA; and (2) polynucleotides that encode wild-type PSEN1 and/or PSEN 2 to replace the corresponding silenced forms of those proteins, as well as methods that utilize such compositions for treatment of neurodegenerative disorders such as Alzheimer's disease.

Polynucleotides

In certain embodiments, each of the antisense oligonucleotide and the wild-type PSEN1 and/or PSEN2 are encoded by polynucleotides. Polynucleotides encoding antisense oligonucleotides are typically shorter in length than polypeptides encoding wild-type PSEN1 and/or PSEN2 and can be synthesized in the laboratory, for example, using an automatic synthesizer, created from other, pre-existing polynucleotides using standard molecular biology and cloning techniques, or a combination of both synthesis and cloning. Polynucleotide encoding wild-type PSEN1 and/or PSEN2 and can also be synthesized in the laboratory, for example, using an automatic synthesizer, created from other, pre-existing polynucleotides using standard molecular biology and cloning techniques, obtained from nucleic acid sequences present in, for example, a mammal such as a human (e.g., as a genomic fragment or as a cDNA reverse-transcribed from a naturally occurring or synthetic mRNA), or any combination of the foregoing. Moreover, any desired changes (i.e., codon modification) in a polynucleotide originally obtained or created from a natural source can be obtained by standard molecular biological techniques such as site-directed mutagenesis or removal and replacement of a portion of the original polynucleotide. One of ordinary skill in the molecular biology art can create the polynucleotides utilized in the present invention without undue experimentation using standard tools and protocols.

The polynucleotides of the present disclosure can be isolated prior to use or insertion into an expression cassette and/or vector. An isolated polynucleotide includes a naturally-occurring polynucleotide that is not immediately contiguous with one or both of the 5′ and 3′ flanking genomic sequences that it is naturally associated with. An isolated polynucleotide can be, for example, a recombinant DNA molecule of any length, provided that the nucleic acid sequences naturally found immediately flanking the recombinant DNA molecule in a naturally-occurring genome is removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules.

Unless otherwise indicated, the term polynucleotide or gene includes reference to the specified sequence as well as the complementary sequence thereof.

Antisense Oligonucleotides

The antisense oligonucleotides utilized in the present expression cassettes, vectors, and methods disclosed herein are designed to hybridize to and prevent expression of endogenous PSEN1 or PSEN2 mRNA. As stated above, endogenous PSEN1 or PSEN2 mRNA includes both wild-type forms and naturally occurring mutant forms. It will be readily apparent to those of skill in the art that an antisense portion of an antisense oligonucleotide that is perfectly complementary to a target region of wild-type PSEN1 mRNA will necessarily have one or more mismatches to a mutant PSEN1 mRNA having mutation(s) that occur in the target region. While up to 4 mismatches can be tolerated and still cause reduced expression of a target mRNA, perfect complementarity increases the chance of full inhibition of mRNA expression. For this reason, in some embodiments, at least one antisense oligonucleotides has an antisense region that has perfect complementarity to a portion wild-type PSEN1 mRNA; and at least one antisense oligonucleotides has an antisense region that has perfect complementarity to a portion of the mutant PSEN1 mRNA present in a subject to whom the antisense oligonucleotide will be delivered. It should be understood that if an antisense oligonucleotide targets a region of PSEN1 mRNA which is not endogenously mutated, the antisense portion of that antisense oligonucleotide will have perfect complementarity to the corresponding region of both the wild-type and mutant form found in a subject. If the antisense portion of an antisense oligonucleotide targets a region of PSEN1 mRNA which comprises a mutation, then two or more antisense oligonucleotides, each targeting different regions of the PSEN1 mRNA, must be employed to obtain perfect complementarity with both the wild-type and mutant PSEN1 mRNAs. In some embodiments, two or more antisense oligonucleotides are employed even if one is capable of perfect complementarity with both wild-type and mutant PSEN1 mRNAs.

In some embodiments, the antisense oligonucleotides of the disclosure are encoded by a polynucleotide that is expressed in a subject (i.e., using gene therapy). In such embodiments, the antisense oligonucleotides are produced by expression of a DNA polynucleotide encoding the antisense oligonucleotide, which is present on a vector administered to a subject. Such encoded antisense oligonucleotides include shRNAs, and miRNAs.

In some embodiments, the antisense oligonucleotides of the disclosure are created ex vivo and administered directly to a subject. Method for direct delivery of such oligonucleotides are known in the art and include the use of lipid-based nanoparticles (i.e., liposomes, solid lipid nanoparticles, nanostructure lipid carriers), polymer-based delivery systems (i.e., cationic polymers such as natural DNA-binding proteins, synthetic polypeptides, poly-ethylenimine, and carbohydrate-based polymers such as chitosan), lipid-polymer hybrid nanoparticles, exosomes, and high-density lipoproteins. Such directly administered antisense oligonucleotides include dsRNAs, miRNAs, dsRNA, external guide sequence (EGS), alternate splicers, and any antisense oligonucleotide comprising one or more non-natural nucleobases. Examples of such directly delivered antisense oligonucleotides that target PSEN1 mRNA are those that comprise an RNA sequence encoded by any one of: a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:47, nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, or nucleotides 448-529 of SEQ ID NO:71; b) a modified version of any of the foregoing SEQ ID NOs, wherein the modification is 1, 2, 3, or 4 nucleotide changes; or c) a 19-21 base nucleotide sequence comprising Tor more consecutive bases taken from the 5′ or 3′ end of any of the foregoing SEQ ID NOs or the modified version thereof, wherein the 19-21 base nucleotide sequence comprises no more than 4 mismatches with a corresponding portion of an endogenous PSEN1 mRNA. Examples of such directly delivered antisense oligonucleotides that target PSEN2 mRNA are those that comprise an RNA sequence encoded by any one of: a) SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO: 36, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, or nucleotides 448-529 of SEQ ID NO:78; b) a modified version of any of the foregoing SEQ ID NOs, wherein the modification is 1, 2, 3, or 4 nucleotide changes; or c) a 19-21 base nucleotide sequence comprising 7 or more consecutive bases taken from the 5′ or 3′ end of any of the foregoing SEQ ID NOs or the modified version thereof, wherein the 19-21 base nucleotide sequence comprises no more than 4 mismatches with a corresponding portion of an endogenous PSEN1 mRNA.

RNA interference (RNAi) induces gene silencing by targeting complementary mRNA for degradation. The first step of RNAi involves processing and cleavage of longer double-stranded RNA into siRNAs, generally bearing a 2 nucleotide overhang on the 3′ end of each strand. The enzyme responsible for this processing is an RNase III-like enzyme termed Dicer. When formed, siRNAs are bound by a multiprotein component complex referred to as RISC (RNA-induced silencing complex). Within the RISC complex, siRNA strands are separated and the strand with the more stable 5′-end, termed the guide strand, is typically integrated to the active RISC complex. The loading into RISC is asymmetric and the less thermodynamically stable strand or “passenger strand” is discarded. The guide strand is desirably the antisense strand and various strategies discussed both in the application and known in the art may be employed to favor the antisense strand being selected as the guide strand. The single-stranded siRNA guide strand then guides and aligns the RISC complex on the target mRNA and through the action of catalytic RISC protein, a member of the argonaute family (Ago2), mRNA is cleaved (Dana H, Chalbatani G M, Mahmoodzadeh H, et al. Molecular Mechanisms and Biological Functions of siRNA. Int J Biomed Sci. 2017; 13(2).48-57).

A modulator of expression, function and/or stability of the endogenous PSEN1, PSEN2 or mutants of PSEN1 or PSEN2, can be a double-stranded RNA molecule for use in RNA interference, for example a shRNA or a miRNA. RNA interference (RNAi) is a process of sequence-specific gene silencing by post-transcriptional RNA degradation or silencing (prevention of translation). RNAi is initiated by use of double-stranded RNA (dsRNA) that is homologous in sequence to the target gene to be silenced. A suitable double-stranded RNA (dsRNA) for RNAi contains sense and antisense strands of about 21 contiguous nucleotides corresponding to the gene to be targeted that form 19 RNA base pairs, leaving overhangs of two nucleotides at each 3′ end (Elbashir et al., Nature 411:494-498 (2001); Bass, Nature 411:428-429 (2001); Zamore, Nat. Struct. Biol. 8:746-750 (2001)). dsRNAs of about 25-30 nucleotides have also been used successfully for RNAi (Karabinos et al., Proc. Natl. Acad. Sci. USA 98:7863-7868 (2001). dsRNA also can be synthesized in vitro and introduced into a cell by methods known in the art.

In some embodiments, an siRNA molecule of the present disclosure comprises a sense strand and a complementary, anti-sense strand in which both strands are hybridized together to form a duplex structure and where the start site of the hybridization to the PSEN1 mRNA is between nucleotide 1 to 5999 on the mRNA sequence (which corresponds to the GenBank NM_000021.4 cDNA sequence).

In certain embodiments, an siRNA molecule of the present disclosure comprises a sense strand and a complementary anti-sense strand in which both strands are hybridized together to form a duplex structure and where the start site of the hybridization is between nucleotide 1 to 2230 on the PSEN2 mRNA sequence (GenBank NM_000447).

In some embodiments, the antisense oligonucleotide comprises: ribonucleic acids (RNA), deoxyribonucleic acids (DNA), synthetic RNA or DNA sequences, modified RNA or DNA sequences, complementary DNA (cDNA), short guide RNA (sgRNA), a short interfering RNA (dsRNA), double-stranded DNA (dsDNA), a micro interfering RNA (miRNA), a small, temporal RNA (stRNA), a short hairpin RNA (shRNA), mRNA, nucleic acid sequences comprising one or more modified nucleobases or backbones, or combinations thereof. Another example of an antisense molecule is a double-stranded small interfering RNA (siRNA) or endoribonuclease-prepared siRNA (esiRNA). An esiRNA is a mixture of siRNA oligonucleotides resulting from cleavage of a long double-stranded RNA (dsRNA) with an endoribonuclease such as Escherichia coli RNase III or dicer. esiRNAs are an alternative concept to the usage of chemically synthesized siRNA for RNA Interference (RNAi). An esiRNAs is the enzymatic digestion of a long double stranded RNA in vitro.

Any method or combination of methods can be used to reduce expression of a gene or protein, including knockdown by techniques such as siRNA and antisense oligonucleotides, for example. Silencing polynucleotide molecules such as dsRNA, dsDNA or oligonucleotides of the present disclosure can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional RNA synthesizer. Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK).

In some embodiments, the antisense oligonucleotide is a siRNA or a precursor to a siRNA (e.g., a shRNA or a miRNA). An siRNA is double-stranded RNA molecule having a polynucleotide sense strand and a polynucleotide antisense strand. Each strand of the siRNA molecule is from 15 to 30 nucleotides in length. At least 15 nucleotides of the antisense strand (not all of which need be consecutive) should base pair with a portion of endogenous PSEN1 or PSEN2 mRNA. At least a portion of the sense strand is complementary to at least a portion of the antisense strand, and the siRNA molecule has a duplex region of from 15 to 30 nucleotides in length (not all of which need to be consecutive). In some aspects of these embodiments, the duplex region of an siRNA is 19-27 base pairs in length (e.g, 19-21 base pairs, e.g., 19 base pairs) with an additional two nucleotide 3′ overhang on each strand. In some aspects of these embodiments, the first nucleotide in the antisense strand is uracil (U). In some aspects of these embodiments, nucleotides 2-8 of the antisense strand have perfect complementarity to a portion of PSEN1 or PSEN2 mRNA. In some aspects of these embodiments, the antisense strand will have 1, 2, 3 or 4 mismatches with the PSEN1 or PSEN2 mRNA it targets. In some aspects of these embodiments, those mismatches are located at up to four of nucleotides 1, 10, 11, and 17-21 of the antisense strand. The antisense strand may also have up to 4 mismatches with the sense strand. This facilitates the in vivo unpairing of the duplex formed between the sense and antisense strand, releasing the antisense strand and enabling it to hybridize to the PSEN1 or PSEN2 mRNA. The design of siRNA molecules and the location of potential mismatches with target mRNA are disclosed in P Angart et al., Pharmaceuticals 2013, 6, pp. 440-68, the disclosure of which is herein incorporated by reference.

In some embodiments the antisense oligonucleotides may be isolated. In another embodiment, the antisense oligonucleotides may be recombinant, synthetic and/or modified, or in any other way non-natural or not a product of nature. As described above, the antisense oligonucleotides of the invention may be modified by use of non-natural nucleotides, or may be conjugated to another chemical moiety. For example, such chemical moieties may be a heterologous nucleic acid conferring increased stability or cell/nucleus penetration or targeting, or may be a non-nucleic acid chemical moiety conferring such properties, of may be a label.

Any nucleotide within an antisense oligonucleotide can be modified by including substituents coupled thereto, such as in a 2′ modification. An antisense oligonucleotide can be modified with a diverse group of small molecules and/or conjugates. The antisense oligonucleotides of the disclosure, e.g. dsRNA and dsDNA, may comprise modified nucleotides such as locked nucleic acids (LNAs). The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon. The bridge “locks” the ribose in the 3′-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such oligomers are synthesized chemically and are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the hybridization properties (melting temperature) of oligonucleotides.

In certain embodiments, the antisense oligonucleotide is a shRNA or miRNA. In certain embodiments, the antisense oligonucleotide is a shRNA or miRNA targeting either a coding region or a non-coding region of an mRNA translated from a human wild-type or mutant presenilin 1. In certain embodiments, the antisense oligonucleotide is a shRNA or miRNA targeting either a coding region or a non-coding region of an mRNA translated from a human wild-type or mutant presenilin 2.

As an example, knockdown by siRNAs derived from shRNAs or miRNAs can be combined with any other method to reduce gene or protein expression by a desired amount. In some embodiments, expression of endogenous PSEN1 is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% as compared to expression of the endogenous PSEN1 in untreated cells. In some aspects of these embodiments, expression of endogenous PSEN1 is reduced by at least 50%. In some aspects of these embodiments, expression of endogenous PSEN1 is reduced by at least 90%. In some aspects of these embodiments, endogenous PSEN1 expression (wild type and any mutant forms) is completely eliminated by the derived siRNAs.

In some embodiments, expression of endogenous PSEN2 is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% as compared to expression of the endogenous PSEN2 in untreated cells. In some aspects of these embodiments, endogenous PSEN1 expression (wild type and any mutant forms) is completely eliminated by the derived siRNAs.

Short Hairpin RNAs (shRNAs). In certain embodiments, the antisense oligonucleotide is a short hairpin RNA (shRNA). Short hairpin RNAs comprise an antisense portion, a substantially complementary sense portion and a short spacer in between that forms a loop between the duplex that forms between the substantially complementary antisense and sense strands. The loop (or hairpin) is recognized and cleaved in vivo by Dicer to generate a double stranded siRNA molecule.

Micro-RNA's. In some embodiments, therapeutic compositions and methods described herein take advantage of the miRNA pathway by altering the seed sequence of natural pri-miRNA or pre-miRNA clusters to target the endogenous PSEN1 or PSEN2 mRNA. The hairpin containing pri-miRNAs are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to yield —70 nucleotides pre-miRNA and 21-23 nucleotides miRNAs respectively. The pre-miRNA is transported to the cytoplasm via Exportin-5 and further processed by Dicer to produce a short, partially double-stranded siRNA, in which one strand comprises the antisense portion and is preferably used as the miRNA guide strand.

In certain embodiments, the silencing polynucleotide is a micro-RNA (miRNA) or pre-micro-RNA (pre-miRNA) both referred as miRNA throughout this application. In some embodiments, the first polynucleotide encodes one, two or three miRNAs or pre-miRNAs to suppress expression of PSEN1, PSEN2 or the combination thereof. Pre-miRNAs and miRNAs contains a 19-25 nucleotide long RNA sequence that binds to complementary sequences in PSEN1 or PSEN2 mRNA and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. A miRNA or pre-miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-7 at the 5′ end of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the PSEN1 or PSEN2 mRNA target sequence. A miRNA or pre-miRNA will also have additional nucleotides that base pair with the PSEN1 or PSEN2 mRNA target sequence. miRNA mediated down-regulation of gene expression may be caused by cleavage of the target mRNAs, translational inhibition of the target mRNAs, or mRNA decay. miRNA targeting sequences are usually located in the 3′-UTR of the target mRNAs. An endogenous PSEN1 or PSEN2 mRNA may be targeted by more than one miRNAs. In some aspects of these embodiments, the polynucleotide encoding one or more miRNAs or pre-miRNAs is located within an intron of a polynucleotide sequence or an expression cassette.

In some embodiments, therapeutic compositions and methods described herein take advantage of the miRNA pathway by altering the seed sequence of natural miRNAs to target the endogenous PSEN1 or PSEN2 genes. In one embodiment, the shRNA or miRNA targeting the PSEN1 or PSNE2 mRNA comprise a miRNA seed match for the guide strand. In another embodiment, the siRNA duplexes or encoded dsRNA targeting the PSEN1 or PSNE2 mRNA comprise a miRNA seed match for the passenger strand.

In one embodiment, portion of the 3′ stem arm of the shRNA or miRNA targeting the PSEN1 or PSEN2 mRNA may have partial complementarity to portion of the passenger strand in the 5′ stem arm.

In one embodiment, the antisense strand of the shRNA or miRNA biding to Dicer and targeting the PSEN1 or PSEN2 mRNA will be more highly favored as the guide strand as compared to the sense strand (which will be favored to be the passenger strand). In one embodiment, the sense strand portion of a shRNA or miRNA is engineered with 1, 2, 3 or 4 mismatches to the antisense portion in order to favor antisense strand loading into RISC as the guide strand.

A shRNA or miRNA is an RNA molecule having a first region, a loop or hairpin region, and a second region. The first and second region can be substantially complementary to each other. In some embodiments, the first and second region are perfectly complementary to each other. Thus, shRNAs and miRNAs can have a stem-loop structure. As used herein the terms “complementary” and “complementarity” are meant to refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotides in anti-parallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art appreciate, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine.

Perfect complementarity or 100% complementarity refers to a situation in which each nucleotide of one polynucleotide strand can hydrogen bond with a nucleotide of an anti-parallel polynucleotide strand. Less than perfect complementarity refers to a situation in which some, but not all, nucleotides of two strands can hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. As another example, if 18 nucleotides out of 20 nucleotides on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 90% complementarity. “Substantial complementarity” refers to polynucleotide strands exhibiting 79% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected to be non-complementary. Accordingly, complementarity does not consider overhangs that are selected to not be similar or complementary to the nucleotides on the anti-parallel strand, unless context clearly indicates otherwise.

The loop of an shRNA and miRNAs can be about 4 to 30 nucleotides in length. In some embodiments, the loop can be between about 4 and about 15 nucleotides in length. The first and the second region can be between about 19 and about 35 nucleotides in length. In some embodiments, the first and the second region are 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides in length. The first and the second region can be of the same length or can be of different lengths. The lengths of the first and the second region can differ by 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, or more. Differences in length can appear as a bulge or as an overhang.

A shRNA and miRNA can be organized in a 5′-antisense-loop-sense-3′ fashion or in a 5′-sense-loop-antisense-3′ fashion. As used herein, the term “antisense strand” refers to a polynucleotide or region of a polynucleotide that is at least substantially (e.g., about 80% or more) complementary to a target nucleic acid of interest. The antisense strand can be about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, and any number or range in between, complementary to a target nucleic acid of interest. Similarly, an antisense strand of a dsRNA can be at least substantially complementary to its sense strand.

The shRNA and miRNA antisense oligonucleotides can include nucleotides in addition to the antisense region, sense region and loop or linker region. For example, these antisense oligonucleotides can also contain overhang nucleotides and additional stem nucleotides that are complementary to other stem nucleotides, but not complementary to the target. The antisense and sense regions of a shRNA or miRNA can include mismatches (i.e., are not perfectly complementary). For example, a sense and an antisense region can have 1 mismatch, 2 mismatches, 3 mismatches, 4 mismatches, 5 mismatches, or more mismatches. Mismatches can be contiguous or can be located anywhere along the sense and antisense regions. Mismatches between a sense and antisense region can result in a bulge. In some embodiments, an antisense region can have perfect complementarity to the sense region. In some embodiments, and antisense and sense region of a shRNA or miRNA have at

The degree of complementarity between the antisense portion of the shRNA or miRNA and the target region of the PSEN1 or PSEN2 mRNA is important in determining the degree to which that mRNA is silenced. In certain embodiments, the antisense portion of the shRNA or miRNA is perfectly complementary to a portion of the PSEN1 or PSEN2 mRNA. This typically results in degradation of the PSEN1 or PSEN2 mRNA with no endogenous protein production. In certain embodiments, the mRNA binding portion of the shRNA or miRNA comprises 1, 2, 3 or 4 mismatches with the target region of PSEN1 or PSEN2 mRNA. One or more mismatches between an antisense region and a target mRNA can result in translational repression rather than degradation of the target mRNA. The mRNA binding targets can be in any region of the PSEN1 or PSEN2 mRNA. In certain embodiments, the sequence targeted by shRNA comprises a GC content from about 30% to about 50% GC. In certain embodiments, the targeted sequence comprises 4 or less consecutive T residues. It should be understood that in a shRNA or miRNA the antisense region can have perfect complementarity to the sense region, but have 1, 2, 3, or 4 mismatches with respect to the target mRNA. Similarly, the antisense region can have mismatches with the sense region of a shRNA or miRNA, while the antisense region has prefect complementarity to the target mRNA.

In some embodiments, therapeutic compositions and methods described herein take advantage of combining 1, 2, 3, 4, 5 or 6 pri-miRNAs or pre-miRNAs under the same promoter to target the endogenous PSEN1 or PSEN2 mRNA at various sites. The target site sequence may comprise a total of 5-100, or more nucleotides, which need not be contiguous.

Expression of shRNAs can be driven by a RNA pol II or III promoter. Exemplary RNA pol III promoters include a U6 promoter, a U61 promoter, a U69 promoter, a H1 promoter, and others. Transcription from a RNA pol III promoter can terminate at a poly T stretch, such as 5 Ts or 6 Ts, for example. shRNAs can also be expressed using a RNA pol II promoter. Use of an RNA pol II promoter can allow for specific and inducible expression, for example.

In certain embodiments, the first polynucleotide encoding a shRNA or miRNA comprises a sequence set forth in any one of SEQ ID NOs:1-36 or 44-47. In some embodiments, the first polynucleotide encoding a shRNA or miRNA comprises a sequence that has 1, 2, 3 or 4 different nucleotides in the antisense region as compared to any one of SEQ ID NOs:1-36 or 44-47.

In certain embodiments, the first polynucleotide encoding a shRNA or miRNA comprises a nucleotides 497-517 of SEQ ID NO:68, nucleotides 497-517 of SEQ ID NO:69, nucleotides 497-517 of SEQ ID NO:70, nucleotides 497-517 of SEQ ID NO:71, nucleotides 497-517 of SEQ ID NO:76, nucleotides 497-517 of SEQ ID NO:77, or nucleotides 497-517 of SEQ ID NO:78. In some embodiments, the first polynucleotide encoding a shRNA or miRNA comprises a sequence that has 1, 2, 3 or 4 different nucleotides in the antisense region as compared to any one of nucleotides 497-517 of SEQ ID NO:68, nucleotides 497-517 of SEQ ID NO:69, nucleotides 497-517 of SEQ ID NO:70, nucleotides 497-517 of SEQ ID NO:71, nucleotides 497-517 of SEQ ID NO:76, nucleotides 497-517 of SEQ ID NO:77, or nucleotides 497-517 of SEQ ID NO:78.

In certain embodiments, the first polynucleotide encoding a shRNA or miRNA comprises a nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, nucleotides 448-529 of SEQ ID NO:71, nucleotides 448-529 of SEQ ID NO:76, nucleotides 448-529 of SEQ ID NO:77, or nucleotides 448-529 of SEQ ID NO:78. In some embodiments, the first polynucleotide encoding a shRNA or miRNA comprises a sequence that has 1, 2, 3 or 4 different nucleotides in the antisense region as compared to any one of nucleotides 448-529 of SEQ ID NO:68, nucleotides 448-529 of SEQ ID NO:69, nucleotides 448-529 of SEQ ID NO:70, nucleotides 448-529 of SEQ ID NO:71, nucleotides 497-517 of SEQ ID NO:76, nucleotides 497-517 of SEQ ID NO:77, or nucleotides 497-517 of SEQ ID NO:78.

It should be understood by those of skill in the art, that in some embodiments, the shRNA or miRNA will be encoded on the same vector that encodes the replacement PSEN1 or PSEN2. The location of the shRNA or miRNA target encoding sequences in that vector can vary (e.g., they can be located 5′ or 3′ to the sequence encoding the replacement PSEN1 or PSEN2), as long as it does not disrupt the expression of the replacement PSEN1 or PSEN2. Multiple copies of the sequences encoding the shRNA or miRNA target sequences may be utilized (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies). When multiple copies are present, they may be located in tandem or placed at different locations with respect to the encoded PSEN1 or PSEN2 replacement sequence. When miRNA target encoding sequences are utilized, they may encode targeting sequences for a single miRNA or multiple miRNAs (e.g., 2, 3, 4 or 5 different miRNAs). Thus, in some embodiments, when miRNA target encoding sequences encoding targeting sequences for multiple miRNAs are utilized, 1, 2, 3, 4, or 5 copies of each specific miRNA target encoding sequences may be used.

Codon Modification

The polynucleotides encoding replacement PSEN1 or PSEN2 can be modified to prevent targeting of the mRNA transcribed therefrom by antisense oligonucleotides targeting endogenous PSEN1 or PSEN2. This can prevent mRNA degradation and RNA silencing or knockdown of the replacement PSEN1 or PSEN2 coding sequence that would otherwise occur. The redundancy of the genetic code can be used to change codons in a target sequence for antisense oligonucleotides, while preserving the amino acid sequence of the protein expressed from the replacement coding sequence.

An endogenous PSEN1 or PSEN2 mRNA being targeted can have mutations that result in the generation of a mutated protein. One or both alleles of an endogenous PSEN1 or PSEN2 gene can be mutated in a subject. In an embodiment, one allele of an endogenous PSEN1 or PSEN2 gene is wild-type and one allele is mutated. In another embodiment, both alleles are mutated. Any mutation can be present in an endogenous allele, including point mutations, substitutions, insertions, deletions, inversions, missense mutations, nonsense mutations, frameshift mutations, translocations, and others. Mutations can be single nucleotide changes (e.g., one or more point mutation) or can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide changes.

The mutation of an endogenous allele can be a dominant negative mutation. A dominant negative mutation can contribute to development of a disease, disorder, or condition or can contribute to susceptibility to a disease, disorder, or condition. In an embodiment, an endogenous PSEN1 gene is mutated in a subject. Dominant negative mutations of a PSEN1 gene can increase susceptibility to Alzheimer's disease by inhibiting the assembly and function of the gamma secretase, for example. A codon-modified or non-codon modified polynucleotide cDNA encoding PSEN1 can be used to restore wild-type PSEN1 expression. Simultaneously endogenous mutated PSEN1 expression can be reduced by targeting the coding regions or non-coding regions of the endogenous PSEN1 mRNA using one or more small RNAs, for example, one or more shRNAs. In an embodiment, the small RNA is an siRNA derived from a shRNA. In certain embodiments, the PSEN1 gene, the PSEN2 gene or the combination thereof, comprise one or more mutations. A codon-modified or non-codon modified polynucleotide cDNA encoding PSEN1, PSEN2 can be used to restore wild-type PSEN1, PSEN2 or the combination thereof, expression.

Alzheimer's disease (AD) patients with an inherited form of the disease carry mutations in the presenilin proteins (PSEN1-UniProtKB-P49768; PSEN2-UniProtKB -P49810) or in the amyloid precursor protein (APP). These disease-linked mutations result in increased production of the longer form of amyloid-beta (main component of amyloid deposits found in AD brains). AD typically begins with subtle memory failure that becomes more severe and is eventually incapacitating. Other common findings include confusion, poor judgment, language disturbance, agitation, withdrawal, hallucinations, seizures, Parkinsonian features, increased muscle tone, myoclonus, incontinence, and mutism. Familial AD (FAD) characterizes families that have more than one member with AD and usually implies multiple affected persons in more than one generation. Early-onset FAD (EOFAD) refers to families in which onset is consistently before age 60 to 65 years and often before age 55 years. The three clinically indistinguishable subtypes of EOFAD based on the underlying genetic mechanism are: Alzheimer disease type 1 (AD1), caused by mutation of APP (10%-15% of EOFAD); Alzheimer disease type 3 (AD3), caused by mutation of PSEN1, (30%-70% of EOFAD); and Alzheimer disease type 4 (AD4), caused by mutation of PSEN2 (<5% of EOFAD). Presenilins are postulated to regulate APP processing through their gamma-secretase function, an enzyme that cleaves APP. Also, it is thought that the presenilins are involved in the cleavage of the Notch receptor, such that they either directly regulate gamma-secretase activity or themselves are protease enzymes.

Mutated PSEN1s in subjects with early onset of Alzheimer's disease, have been found to include mutations such as substitutions, insertions (ins), deletions (del), inversions, missense, frameshift (fs), exon deletions (Δ) and the like. Examples of such amino acid changes throughout the PSEN1 protein include: Q15H, N32N, R35Q, N39Y, D4Odel (delGAC), D4Odel (delACG), R42L, E69D, A79V, V82L, I83_M84del (DelIM, ΔI83/M84, ΔI83/ΔM84), I83T, M84T, M84V, L85P, P88H, P88L, V89L (G>C), V89L (G>T), C92S, V94M, V96F, V97L, T99A, F105C, F105I, F105L, F105V, R108Q, G111V, G111W, L113_I114insT, L113P, L113Q, Y115C, Y115D, Y115H, T116I, T116S, P117T, T116N, T116R, P117A, P117L, P117Q, P117R, P117S, T119I, E120D (A>C), E120D (A>T), E120G, E120K, T122A, E123K, H131R, S132A, L134R, N135D, N135S, N135Y, A136G, A137T, M139I (G>C), M139I (G>A), M139K, M139L, M139T, M139V, V142F, V142I, I143F, I143M, I143N, I143T, I143V, M146I (G>T), M146I (G>C), M146I (G>A), M146L (A>C), M146L (A>T), M146V, T147I, T147P, L150P, L153V, Y154C, Y154N, Y156F, Y156 R157insIY, R157S, Y159F, H163P, H163R, H163Y, A164V, W165C (G>T), W165C (G>C), W165G, L166H, L166P, L166R, L166V, L166del, I167del (TTAdel), I167del (TATdel), I168T, S169del (ΔS169, Ser169del, ΔS170), S169L, S169P, S170F, S170P, L171P, L173F (G>T), L173F (G>C), L173S, L173W, L174del, L174M, L174R, F175del, F175S, F176L, F177L, F177S, S178P, I180N, G183V, E184D, E184G, V191A. I202F, W203C, F205_G206del;insC, G206A, G206D, G206S, G206V, G209A, G209E, G209R, G209V, M210R, S212Y, I213F, I213L, I213T, H214D, H214N, H214R, H214Y, G217D, G217R, L219F, L219P, L219R, R220P, Q222H, Q222P, Q222R, Q223R, L226F, L226R, I227V, I229F, S230I, S230N, S230R, A231P, A231T, A231V, L232P, M233I (G>A), M233I (G>C), M233L (A>C), M233L (A>T), M233T, M233V, L235P, L235R, L235V, F237C, F237I, F237L, I238M, K239N, L241R, T245P, A246E, A246P, L248P, L248R, I249L, L250F, L250S, L250V, Y256N, Y256S, A260V, V261F, V261I, V261L, L262F, L262S, L262V, C263F, C263R, P264L, G266S, P267A, P267L, R269G, R269H, L271V, V272A, V272D, E273A, E273G, T274R, A275V, R278I, R278K, R278S, R278T, E280A, E280G, E280K, L282F, L282R, L282V, F283L, P284L, P284S, A285V, L286P, L286V, T291A, T291P, P303L, K311R, E318G, D333G, R352C, R352 S353insR, T354I, R358Q, A360T, S365A, S365Y, R377M, R377W, G378E, G378V, G378fs, L381F, L381V, G384A, F386I, F386L, F386S, F388L, S390I, S390N, V391F, V391G, L392P, L392V, V393F, G394V, A396T, N405S, I408T, A409T, C410Y, V412I, I416T, G417A, G417S, L418F, L420R, L424F, L424H, L424P, L424R, L424V, A426P, A431E, A431V, P433S, A434C, A434T, L435F, P436Q, P436S, I437V, I439S, I439V, T440del, 869-2A>G, 869-22_869-23ins18 (4E9, 49, deltaE9), I238_K239insI, L171_L172insY, S290C; T291_S319del (ΔE9, Δ9), S290C; T291_S319del A>G (ΔE9, Δ9), S290C; T291_S319del G>A (ΔE9, Δ9), S290C; T291_S319del G>T (ΔE9, Δ9), S290W; S291_R377del (Δ9-10, Delta9-10, p.Ser290_Arg377delinsTrp, g.73671948_73682054del).

In a genetic screening study of familial and sporadic early-onset Alzheimer disease (EOAD), a censoring effect was observed in families of patients carrying the c.772T>C, p.(Leu241Arg), the c.539T>A, p.(Ile180Asn), and the c.710T>G, p.(Phe237Cys) substitutions, while the c.331G>T, p.(Gly111Trp), the c.350C>A, p.(Pro117Gln), and the c.614_616del, p.(Phe205_Gly206delinsCys) mutations occurred de novo. One patient carried the c.1078G>A p.(Ala360Thr) variant (Lanoiselée H M, Nicolas G, Wallon D, et al. APP, PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: A genetic screening study of familial and sporadic cases. PLoS Med. 2017;14(3):e1002270. Published 2017 Mar 28. doi:10.1371/journal.pmed.1002270). There is a need for screening nonfamilial AD cases and compositions and methods for the treatment of neurodegenerative diseases such as AD, including effective gene and combination therapies.

PSEN2 mutations are associated with variable penetrance and a wide range in the age of disease onset, from 45 to 88 (Bird T D, Levy-Lahad E, Poorkaj P, et al. Ann Neurol. 1996; 40(6):932-936. Sherrington R, Froelich S, Sorbi S, et al. Hum Mol Gen. 1996;5(7):985-988). PSEN2 mutations are associated with both EOAD and late-onset Alzheimer disease (LOAD). Only 17 of the 38 are predicted to be disease-causing mutations. Ten of the mutations are not pathogenic and the others are still unclear. Sixteen mutations are located within transmembrane domains. Cell-based studies suggest that four of these mutations, T122P, N141I, M239I, and M239V, cause an increase in the amount of Aβ peptide. The mutations T122R, S130L, and M239I were found to alter calcium signaling. Most of these mutations were discovered in European and African populations. Until now, only four missense mutations were described in Asian populations: Asn141Tyr was associated with EOAD in a Chinese Han family; Gly34Ser was found in a Japanese patient; and Arg62Cys and Va1214Leu were described in the Korean patients (Yan Cai et al., 2015, vol. 10, pages 1163-1172). Two PSEN2 mutations, Glu126fs and Lys306fs, are frameshift mutations, and the others are nonsynonymous substitutions (Lamer A J. Epilepsy & Behavior. 2011; 21(1):20-22).

In certain embodiments the polynucleotide encoding replacement PSEN1 and/or PSEN2 is codon optimized. Codon optimization is a form of codon modification that can be utilized to enhance protein expression for heterologous gene expression. Codon optimization is a method of gene optimization, wherein the synthetic coding sequence is modified to match the “codon usage pattern” for a particular organism. For example, in order to optimize expression of a particular amino acid sequence in a specific organism, one would select the “most frequently used codons” (from a list of degenerate codons for an amino acid), by that organism. Upon codon optimization, the encoded amino acid sequence remains the same but with the DNA sequence encoding the amino acid sequence is different, optimized for that organism. Optimized codons for PSEN1 and PSEN2 coding sequences are shown in the Table below.

TABLE 1

Preferred Optimized Codons

Amino acid
Most preferred codon
Preferred codon

A (Alanine)
GCC
GCT, GCA

C (Cysteine)

TGT, TGC

D (Aspartate)

GAT, GAC

E (Glutamate)

GAA, GAG

F (Phenylalanine)

TTT, TTC

G (Glycine)
GGC
GGA, GGG

H (Histidine)

CAT, CAC

I (Isoleucine)
ATC
ATT

K (Lysine)

AAA, AAG

L (Leucine)
CTG
TTG, CTT, CTC

M (Methionine)

ATG

N (Asparagine)

AAT, AAC

P (Proline)

CCT, CCC, CCA

Q (Glutamine)
CAG

R (Arginine)
AGA, AGG
CGC, CGG

S (Serine)
AGC
TCT, TCC, TCA, AGT

T (Threonine)
ACC
ACT, ACA

V (Valine)
GTG
GTT, GTC

W (Tryptophan)

TGG

Y (Tyrosine)

TAT, TAC

STOP
TGA
TAA

In some embodiments, the polynucleotide encoding a replacement PSEN1 is nucleotides 1906-3303 of SEQ ID NO:68.

Expression Cassettes

In addition to polynucleotide sequences encoding replacement wild-type PSEN1 and/or PSEN2 that are resistant to silencing by an antisense oligonucleotide (and in certain embodiments, polynucleotide sequences encoding such antisense oligonucleotides), the expression cassettes provided herein may contain certain non-coding regions that are integral to the function of cells, particularly the control of gene activity. These are termed regulatory elements. It should be apparent to those of skill in the art, that some or even all of these non-coding regions may alternatively be provided in the vector into which the expression cassette is inserted. Regardless of the location of these non-coding sequences (expression cassette or vector), they must be operably linked to the polynucleotide sequence encoding the antisense oligonucleotide and the polynucleotide sequence encoding the replacement PSEN1 or PSEN2 coding sequence.

The role of these noncoding sequence varies. For example, noncoding DNA contains sequences that act as regulatory elements, including the transcriptional and translational regulation of protein-coding sequences, origins of DNA replication, centromeres, telomeres, scaffold attachment regions (SARs), genes for functional RNAs. Noncoding DNA contains many types of regulatory elements, such as, for example, promoters, enhancers or silencers which provide binding sites for proteins that repress transcription. Like enhancers, silencers can be found before or after the gene they control or are cis-acting. Insulators provide binding sites for proteins that control transcription in a number of ways. Some prevent enhancers from aiding in transcription (enhancer-blocker insulators). Others prevent structural changes in the DNA that repress gene activity (barrier insulators). Some insulators can function as both an enhancer blocker and a barrier. Non-coding regions can, for example, include a 5′ untranslated region (“UTR”), a 3′ UTR, or both.

An expression cassette can comprise a polynucleotide comprising a PSEN1 or PSEN2 coding sequence and optionally, regulatory elements preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence that are required for expression of the selected gene product. Thus, an expression cassette can comprise: 1) a promoter sequence; 2) an intron 3) a PSEN1 or PSEN2 coding sequence; and, 4) a 3′ untranslated region (i.e., a terminator) that, in eukaryotes, usually contains a polyadenylation site.

Similarly, an expression cassette can comprise a polynucleotide encoding one or more antisense oligonucleotides, e.g., a shRNA, or a miRNA and can comprise regulatory elements preceding (i.e., 5′ to) and following (i.e., 3′ to) the sequence encoding the shRNA or miRNA that are required for expression. Thus, an expression cassette can comprise, for example: 1) a promoter sequence; 2) an intron 3) a sequence encoding one or more shRNAs or miRNAs; and, 4) a 3′ region (i.e., a terminator) that specifies the end of transcription of the RNA. Each shRNA or miRNA can have its own promoter and intron. Alternatively, one promoter can be operably linked to a series of 2, 3, 4, 5, or more shRNAs or miRNAs .

One or more shRNAs or pre-miRNAs can occur in a series that is operably linked to a promoter. Pre-miRNAs or shRNAs occurring in a series means that the pre-miRNAs or shRNAs are arranged together or close together and are all operably linked to one or more 5′ promoters. Accordingly, a first polynucleotide can comprise one or more 5′ promoters driving miRNA or shRNA expression. In an embodiment, a first polynucleotide comprises one or more miRNAs or shRNAs linked to a single 5′ promoter (see, e.g., SEQ ID NO: 37 and SEQ ID NO:38). In another embodiment, a first polynucleotide comprises one or more miRNAs or shRNAs, with each miRNA or shRNA linked to a different 5′ promoter (see, e.g., SEQ ID NO: 49). Any number of promoters can drive expression of any number of miRNAs or shRNAs of a first polynucleotide. For example, a 5′ promoter can drive one or more miRNAs or shRNAs, and another 5′ promoter can drive one or more different miRNAs or shRNAs. Promoters driving expression of different miRNAs or shRNAs or different numbers of miRNAs or shRNAs can be the same or different promoters.

Methods for preparing polynucleotides operably linked to a regulatory element and expressing polypeptides in a host cell are well-known in the art. See, e.g., U.S. Pat. No. 4,366,246. A polynucleotide can be operably linked when it is positioned adjacent to or close to one or more regulatory elements, which direct transcription and/or translation of the polynucleotide.

An expression cassette can be a circular or linear nucleic acid molecule. In some cases, an expression cassette is delivered to cells (e.g., a plurality of different cells or cell types including target cells or cell types and/or non-target cell types) in a vector (e.g., an expression vector).

Regulatory Elements

As stated above, the expression cassettes disclosed herein can include one or more regulatory elements operably linked to a polynucleotide encoding PSEN1 (or PSEN2) or to a polynucleotide encoding an antisense oligonucleotide, such as a shRNA. A regulatory element is a genetic element or polynucleotide that either alone or together with one or more additional regulatory elements influences or modulates expression of a polynucleotide or gene. A regulatory element can facilitate polynucleotide or gene expression, increase polynucleotide or gene expression, decrease polynucleotide or gene expression and/or confer selective polynucleotide or gene expression in a particular cell type or tissue. A regulatory element can influence or modulate polynucleotide or gene expression temporally and/or spatially. As used herein, the term “regulate polynucleotide or gene expression,” “influence polynucleotide or gene expression,” or “modulate polynucleotide or gene expression” refers to increasing polynucleotide or gene expression, decreasing polynucleotide or gene expression, and/or conferring selective polynucleotide or gene expression. “Regulating polynucleotide or gene expression,” “influencing polynucleotide or gene expression,” or “modulating polynucleotide or gene expression” can refer to temporal and/or spatial regulation.

Any genetic element that modulates or influences polynucleotide or gene expression can be a regulatory element, including, for example, promoters, enhancers, chromatin insulators, translation initiation sequences such as strong and weak Kozak signal sequences, internal ribosomal entry sites, mRNA stability sequences, sequences that influence mRNA processing such as splicing and cleavage, sequences that influence mRNA export from the nucleus and/or mRNA retention, posttranslational response elements, non-coding sequences such as introns and untranslated regions (UTRs), poly A sequences, repressors, silencers, terminators, and others. Regulatory elements can function to modulate polynucleotide or gene expression at the transcriptional level, at the posttranscriptional level, at the translational level, or any combination thereof. Regulatory elements can increase the rate at which RNA transcripts are produced, increase the stability of RNA produced, increase the rate of protein synthesis from RNA transcripts, prevent RNA degradation and/or increase RNA stability to facilitate protein synthesis, for example. Regulatory elements can be located in an inverted terminal repeat (ITR) sequence or a long terminal repeat (LTR).

Nucleic acid expression cassettes described herein can comprise regulatory elements that regulate or modulate polynucleotide or gene expression at any step, including the transcriptional, posttranscriptional, and translational levels, for example. A regulatory element can regulate or modulate polynucleotide or gene expression at more than one level or function in more than one way to regulate or modulate polynucleotide or gene expression. Thus, a regulatory element can have any function or any combination of the functions described above. For example, a regulatory element can function as an mRNA stabilizing element and modulate, i.e., increase or decrease, translation. As yet another example, a regulatory element can modulate transcription initiation and modulate mRNA stability. A regulatory element can also have a predominant function by which it modulates polynucleotide or gene expression and have one or more additional functions that increase or decrease polynucleotide or gene expression. A regulatory element can comprise a sequence that is located within or overlaps with other regulatory elements that have the same or different functions in modulating polynucleotide or gene expression or that modulate polynucleotide or gene expression at the same or different steps.

Regulatory elements can be derived from coding or non-coding DNA sequences. Regulatory elements derived from non-coding DNA can be associated with genes, e.g., can be found in a gene, such as upstream sequences, introns, 3′ and 5′ untranslated regions (UTRs), and/or downstream regions. As used herein, the term “upstream” when referring to nucleic acid means 5′ relative to another sequence and the term “downstream” means 3′ relative to another sequence. The term “upstream” can be used interchangeably with the term “5′” when referring to location of sequences relative to each other, unless context clearly indicates otherwise. The term “downstream” can be used interchangeably with the term “3′” when referring to location of sequences relative to each other, unless context clearly indicates otherwise.

In some embodiments, regulatory elements derived from non-coding DNA sequences are not associated with a gene, e.g., may not be found in a gene. The genomic region from which a regulatory element is derived can be distinct from the genomic region from which an operably linked polynucleotide is derived. In some embodiments, a regulatory element is derived from a distal genomic region or location with respect to the genomic region or location from which the operably linked polynucleotide (such as a cDNA derived from an endogenous gene or an endogenous version of a heterologous gene, for example) is derived. In some embodiments, a regulatory element comprises intron sequences. Intron sequences can include sequences derived from any gene. In some embodiments, the intron sequences are derived from the genomic region from which an operatively linked polynucleotide is derived. For example, the nucleic acid expression cassettes described herein can include introns from an endogenous gene that corresponds to a polynucleotide or that gave rise to a polynucleotide in the form of a cDNA. As another example, nucleic acid expression cassettes described herein can include introns from an endogenous gene that does not correspond to or gave rise to a polynucleotide.

Promoters

A promoter is a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5′ of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell-or tissue-specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Promoters are typically classified into two classes: inducible and constitutive. A constitutive promoter refers to a promoter that allows for continual transcription of the coding sequence or gene under its control.

An inducible promoter refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. If inducible, there are inducer polynucleotides present therein that mediate regulation of expression so that the associated polynucleotide is transcribed only when an inducer molecule is present. A directly inducible promoter refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed. An indirectly inducible promoter refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by inducible promoter.

A promoter can be any polynucleotide that shows transcriptional activity in the chosen host organism (e.g., a mammal such as a human). A promoter can be naturally-occurring, can be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is derived from studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-61 (1987). In addition, the location of the promoter relative to the transcription start can be optimized. Many suitable promoters for use in mammals and mammalian cells are well known in the art, as are polynucleotides that enhance expression of an associated expressible polynucleotide.

Eukaryotic promoters include RNA pol I, RNA pol II, and RNA pol III promoters. RNA pol I can transcribe genes encoding ribosomal RNAs, for example. RNA pol II can transcribe genes encoding mRNAs, small nuclear RNAs, and micro interfering RNAs, for example. RNA pol III can transcribe genes encoding tRNAs, ribosomal RNAs, and other small RNAs, for example. RNA pol II promoters can provide inducible gene expression and selective or tissue-specific gene expression, for example.

A promoter can be a neuron-specific promoter. A neuron-specific promoter can provide selective expression of a polynucleotide or therapeutic gene in neuronal cells. Selective expression that is restricted or limited to a particular cell type can prevent or reduce off-target effects that are often undesirable and can result in side effects, for example. As used herein, “selective expression” refers to expression that is significantly greater (i.e, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold or higher in neurons as compared to non-neuronal cells. In some embodiments, there is no expression in non-neuronal cells. Moreover, when a neuron specific promoter is utilized, the polynucleotides operatively linked thereto can be expressed in at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or in 100%, and any number or range in between, of neurons.

RNA pol II promoters that are selective for a particular cell type or target cell can provide strong expression in the target cell compared to a general promoter that can drive expression in any cell type or compared to a promoter that drives expression in one or more cell types other than the target cell. In some embodiments, a neuron-specific promoter of the nucleic acid expression cassettes described herein provides for expression that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and any number or range in between, higher as compared to expression provided by a promoter that can drive expression in any cell type. In some embodiments, a neuron-specific promoter of the nucleic acid expression cassettes described herein provides for expression that is at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and any number or range in between, higher as compared to expression provided by a promoter that can drive expression in one or more non-neuronal cell types.

Any neuron-specific promoter can be used in the nucleic acid expression cassettes provided herein. Exemplary promoters include the somatostatin (SST) gene promoter SEQ ID NO: 63, the neuropeptide Y (NPY) promoter SEQ ID NO: 62, the alpha-calcium/calmodulin kinase 2A promoter, a synapsin I promoter SEQ ID NO: 64 or SEQ ID NO: 65, neuron-specific enolase (NSE) SEQ ID NO: 56, dopaminergic receptor 1 (Drd1a) promoter, tubulin alpha I promoter, and others. Hybrid promoters can also be used. As used herein, the term “hybrid promoter” refers to a promoter that includes promoter sequences derived from more than one gene. Promoters can be from any species, including human, rhesus macaque, mouse, rat, and chicken, for example.

In alternate aspects of these embodiments, the promoter is selected from CAG (SEQ ID NO: 50), CBA (SEQ ID NO: 51 or nucleotides 941-1213 of SEQ ID NO:68), UBC (SEQ ID NO: 52), PGK (SEQ ID NO: 53), PKC, EF1a (SEQ ID NO: 54), GUSB (SEQ ID NO: 59), CMV (SEQ ID NO: 55), PDGF, desmin, MCK, MeCP2 (SEQ ID NO: 57), GFAP (SEQ ID NO: 58), MBP, RSV (SEQ ID NO: 60), SV40 (SEQ ID NO: 61), or beta-globin (SEQ ID NO:66).

Chromatin Insulator Sequence

A nucleic acid expression cassette can further comprise a chromatin insulator sequence. Packaging of genes into chromatin can render genes inaccessible to the transcription machinery of the cell, resulting in little or no gene expression. Chromatin insulators can protect a sequence from being packed into transcriptionally inactive chromatin. Including a chromatin insulator sequence in a nucleic acid expression cassette can keep a polynucleotide in an accessible state and allow transcription to occur. Any chromatin insulator can be used in the nucleic acid expression cassettes provided herein. Exemplary chromatin insulator sequences include the CTCF insulator, the gypsy insulator, and the β-globin locus. Chromatin insulator sequences from any species can be used, including mammals and non-mammals and vertebrates and non-vertebrates. As an example, a chromatin insulator sequence from the human beta globin locus HS4 can be used. Other examples of chromatin insulator sequences include sequences form chicken and Drosophila.

The nucleic acid expression cassettes described herein can include regulatory elements that function after transcription has occurred. Post-transcriptional regulatory elements can modulate RNA stability and degradation, processing such as splicing and cleavage, and export from the nucleus, for example. Posttranscriptional regulatory elements can also modulate translation by modulating the amount of mRNA available for translation and by modulation translation initiation, for example.

mRNA Stability Elements

A nucleic acid expression cassette can include at least one mRNA stability element. Any mRNA stability element can be included in the nucleic acid expression cassettes. An mRNA stability element can be an expression and nuclear retention element, a 5′ UTR, a 3′ UTR, elements within UTRs, and others. Exemplary mRNA stability elements include the MALAT1 mRNA stability element, NEAT1 stability element, viral expression and nuclear retention elements from the Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV); and equine herpesvirus 2 (EHV2), and woodchuck posttranscriptional regulatory element (WPRE), C-rich stability elements of the HBA1, HBA2, lipoxygenase, alpha(I)-collagen, and the tyrosine hydroxylase 3′ UTRs, for example, AU-rich elements (AREs) of 3′ UTRs, and others. An mRNA stability element can be, for example, an expression and nuclear retention element. An mRNA stability element can prevent or decrease degradation of mRNA. For example, degradation of mRNA can be decreased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, and any number or range in between, when an mRNA stability element is included as compared to a nucleic acid expression cassette that does not include an mRNA stability element. In an embodiment, there is no degradation of mRNA. Any sequence that prevents or decreases degradation of the mRNA can be an mRNA stability element. In some embodiments, an untranslated region (UTR) is an mRNA stability element in the nucleic acid expression cassettes provided herein. A 3′ UTR, a 5′ UTR, or a 3′ UTR and a 5′ UTR can be included in the nucleic acid expression cassettes described herein. In some embodiments, the mRNA stability element is a sequence derived from a non-coding sequence or a UTR.

An mRNA stability element can be placed into any location in a nucleic acid expression cassette. For example, an mRNA stability element can be placed 3′ to the open reading frame of a polynucleotide and before or 5′ of a polyadenylation site. As another example, an mRNA stability element can be placed 5′ to the open reading frame of a polynucleotide and 5′ to a polyadenylation site.

A nucleic acid expression cassette can include untranslated regions (UTRs). Generally, a UTR is found on each side of a coding sequence on an mRNA, i.e., an mRNA generally has a 5′ UTR upstream of the coding sequence and a 3′ UTR or trailer sequence immediately following a stop codon.

A 5′ UTR generally includes sequences that are recognized by the ribosome that allow the ribosome to bind and initiate translation. Exemplary sequences for translation initiation include Kozak initiation signal sequences and internal ribosomal entry sites. As used herein, the terms “Kozak initiation signal sequence,” “Kozak consensus sequence,” and “Kozak sequence” can be used interchangeably, unless context clearly indicates otherwise. A person of skill in the art will appreciate that a Kozak initiation signal sequence can be located in part in the 5′ UTR and include the AUG translation initiation codon itself and the nucleotide immediately following or downstream of the AUG start codon, as described below.

Translation initiation of an mRNA typically occurs at an ATG codon that is recognized by a ribosome. The ATG codon at which translation begins may not be the first ATG start codon present in an mRNA sequence. A motif called a Kozak sequence can direct translation initiation to an ATG codon. The Kozak consensus sequence is defined as 5′-(gcc)gccRccAUGG-3, where the underlined AUG indicates the translation start codon; uppercase letters indicate conserved bases; “R” indicates the presence of a purine, with adenine more frequent; lowercase letters indicate the most common base at a position that can vary; and the sequence (gcc) is of uncertain significance. In addition to these features, other positions and features can contribute to translation initiation. Strong and weak Kozak consensus sequences have been described, with a strong Kozak consensus sequence including the features above that are considered optimal for translation initiation and a weak Kozak consensus sequence including features that deviate or differ from a strong Kozak consensus sequence. The amount of protein synthesized from an mRNA can depend on the strength of the Kozak sequence. For example, a CCACC sequence immediately upstream of an AUG translation initiation codon can increase the rate of translation initiation compared to a sequence that differs from CCACC.

In some embodiments, the nucleic acid expression cassettes provided herein comprise a Kozak translation initiation signal. The Kozak translation initiation signal can be located immediately upstream or 5′ of a translation initiation AUG codon. Any Kozak consensus sequence that is a strong Kozak sequence can be used. In some embodiments, the Kozak translation initiation signal comprises a sequence CCACC. Additional Kozak translation initiation sequences that can be used include GCCACC, CCGCC, CCACG, CCGCG, CCACA, CCGCA, and others. As another example, any sequence of XYRYY can be used, where “X” is C or G, “R” is a purine, and “Y” is C, G, or A.

Transcription Termination Region

A transcription termination region of a recombinant construct or expression cassette is a downstream regulatory region including a stop codon and a transcription terminator sequence. Transcription termination regions that can be used can be homologous to the transcriptional initiation region, can be homologous to the polynucleotide encoding a polypeptide of interest, or can be heterologous (i.e., derived from another source). A transcription termination region can be naturally occurring, or wholly or partially synthetic. 3′ non-coding sequences encoding transcription termination regions may be provided in a recombinant construct or expression construct and may be from the 3′ region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts when utilized in both the same and different genera and species from which they were derived. Termination regions may also be derived from various genes native to the preferred hosts. The termination region is usually selected more for convenience rather than for any particular property.

A 3′ UTR generally plays an important role in translation termination and in post-transcriptional gene expression. For example, regulatory regions in a 3′ UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA. A 3′ UTR can contain binding sites for regulatory proteins and for micro interfering RNAs (miRNAs), for example. miRNA binding can decrease expression of an mRNA by inhibiting translation or causing degradation of the transcript. A 3′ UTR can also have silencer regions which bind to repressor proteins, thereby inhibiting the expression or translation of the mRNA. 3′ UTRs can contain AU-rich elements (AREs). Proteins binding to AREs can affect the stability or decay rate of transcripts in a localized manner or affect translation initiation. Generally, a 3′ UTR contains the sequence AAUAAA that directs addition of several hundred adenine residues called the poly(A) tail to the end of the mRNA transcript. Poly(A) binding protein (PABP) can bind to this tail, contributing to regulation of mRNA translation, stability, and export. For example, poly (A) tail bound PABP interacts with proteins associated with the 5′ end of the transcript, resulting in circularization of the mRNA that promotes translation. A 3′ UTR can also contain sequences that attract proteins to associate the mRNA with the cytoskeleton, transport it to or from the cell nucleus, or perform other types of localization. Sequences within the 3′ UTR and physical characteristics of a 3′ UTR, including its length and secondary structure, can contribute to translation regulation. A 3′ UTR can also include elements that modulate mRNA transcription, thus functioning as a transcriptional regulatory element.

In some embodiments, the nucleic acid expression cassettes described herein include a 5′ UTR sequence, a 3′ UTR sequence, or a 5′ UTR sequence and a 3′ UTR sequence. Any 5′ UTR sequence and any 3′ UTR sequence derived from any gene can be used. Preferably, 5′ UTR and 3′ UTR sequences included in the nucleic acid expression cassettes provided herein are derived from human genes, although 5′ UTR and 3′ UTR sequences can be from any gene and from any organism. In some embodiments, the nucleic acid expression cassettes described herein comprise a 5′ UTR sequence, a 3′ UTR sequence, or a 5′ UTR sequence and a 3′ UTR sequence of a presenilin 1 gene. In some embodiments, the nucleic acid expression cassettes described herein comprise a 5′ UTR sequence, a 3′ UTR sequence, or a 5′ UTR sequence and a 3′ UTR sequence of the human presenilin 1 gene. In some embodiments, the 5′ UTR and 3′ UTR sequences included in the nucleic acid expression cassettes function as mRNA stability elements, although any 5′ UTR and/or 3′ UTR sequence can contribute any other function, including any of the functions described above, to modulate expression of a polynucleotide encoding PSEN1 or other therapeutic gene of a nucleic acid expression cassette provided herein. In some embodiments, a 5′ UTR sequence, a 3′ UTR sequence, or a 5′ UTR sequence and a 3′ UTR sequence function to stabilize mRNA.

In some embodiments, the nucleic acid expression cassettes described herein comprise introns. Introns can promote splicing and enhance nuclear export, for example. Any intron sequences from any gene can be used. In some embodiments, the nucleic acid expression cassettes provided herein include intron sequences derived from a gene other than PSEN1. In some embodiments, the introns allow for alternative splicing to create protein isoforms with variant lengths and additional but overlapping functions. Protein isoforms can also have different cellular functions and properties. Alternative splicing can rearrange intron and exon sequences that are joined to alter the mRNA coding sequence. In some embodiments, the nucleic acid expression cassettes provided herein include intron sequences derived from the PSEN1 gene. For example, the cDNA of a polynucleotide encoding PSEN1 can include one or more intron sequences. The one or more intron sequences can be PSEN1 intron sequences or any other intron sequences. In some embodiments, entire intron sequences are included in the nucleic acid expression cassettes described herein. In some embodiments, partial intron sequences are included in the nucleic acid expression cassettes described herein. In some embodiments, a combination of entire and partial intron sequences are included in the nucleic acid expression cassettes described herein.

Regulatory elements and polynucleotides of the nucleic acid expression cassettes provided herein can be combined in any fashion.

Modified or Mutated Nucleic Acid Sequences

In some embodiments, an antisense oligonucleotide may be modified or derived from a native nucleic acid sequence, for example, by introduction of mutations, deletions, substitutions, modification of nucleobases, backbones and the like. The nucleic acid sequences include dsRNA, dsDNA and oligonucleotides, etc. Examples of some modified nucleic acid sequences envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In some embodiments, modified oligonucleotides comprise those with phosphorothioate backbones and those with heteroatom backbones, CH₂—NH—O—CH₂, CH,—N(CH₃)—O—CH₂[known as a methylene(methylimino) or MMI backbone], CH₂—O—N (CH₃)—CH₂, CH₂—N(CH₃)—N (CH₃)—CH₂and O—N (CH₃)—CH₂—CH₂backbones, wherein the native phosphodiester backbone is represented as O—P—O—CH,). The amide backbones disclosed by De Mesmaeker et al. Acc. Chem. Res. 1995, 28:366-374) are also embodied herein. In some embodiments, the nucleic acid sequences having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506), peptide nucleic acid (PNA) backbone wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. Science 1991, 254, 1497). The nucleic acid sequences may also comprise one or more substituted sugar moieties. The nucleic acid sequences may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

The antisense oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N⁶(6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp75-'7′7; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A “universal” base known in the art, e.g., inosine may be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Examples of other modified nucleobases can be found, for example, in Genes VI, Chapter 9 (“Interpreting the Genetic Code”), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington D.C.). Modified RNA components include the following: 2′-O-methylcytidine; N⁴-methylcytidine; N⁴-2′-O-dimethylcytidine; N⁴-acetylcytidine; 5-methylcytidine; 5,2′-O-dimethylcytidine; 5-hydroxymethylcytidine; 5-formylcytidine; 2′-O-methyl-5-formaylcytidine; 3-methylcytidine; 2-thiocytidine; lysidine; 2′-O-methyluridine; 2-thiouridine; 2-thio-2′-O-methyluridine; 3,2′-O-dimethyluridine; 3-(3-amino-3-carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2′-O-dimethyluridine; 5-methyl-2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine; 5-methoxycarbonylmethyluridine; 5-methoxycarbonylmethyl-2′-O-methyluridine; 5-methoxycarbonylmethyl-2′-thiouridine; 5-carbamoylmethyluridine; 5-carbamoylmethyl-2′-O-methyluridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl) uridinemethyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyl-2′-O-methyl-uridine; 5-carboxymethylaminomethyl-2-thiouridine; dihydrouridine; dihydroribosylthymine; 2′-methyladenosine; 2-methyladenosine; N⁶Nmethyladenosine; N⁶, N⁶-dimethyladenosine; N⁶,2′-O-trimethyladenosine; 2 methylthio-N⁶Nisopentenyladenosine; N⁶-(cis-hydroxyisopentenyl)-adenosine; 2-methylthio-N⁶-(cis-hydroxyisopentenyl)-adenosine; N⁶-glycinylcarbamoyl)adenosine; N⁶threonylcarbamoyl adenosine; N⁶-methyl-N⁶-threonylcarbamoyl adenosine; 2-methylthio-N⁶-methyl-N⁶-threonylcarbamoyl adenosine; N⁶-hydroxynorvalylcarbamoyl adenosine; 2-methylthio-N⁶-hydroxnorvalylcarbamoyl adenosine; 2′-O-ribosyladenosine (phosphate); inosine; 2′O-methyl inosine; 1-methyl inosine; 1,2′-O-dimethyl inosine; 2′-O-methyl guanosine; 1-methyl guanosine; N²-methyl guanosine; N², N²-dimethyl guanosine; N², 2′-O-dimethyl guanosine; N², N², 2′-O-trimethyl guanosine; 2′-O-ribosyl guanosine (phosphate); 7-methyl guanosine; N², 7-dimethyl guanosine; N², N²;7-trimethyl guanosine; wyosine; methylwyosine; under-modified hydroxywybutosine; wybutosine; hydroxywybutosine; peroxywybutosine; queuosine; epoxyqueuosine; galactosyl-queuosine; mannosyl-queuosine; 7-cyano-7-deazaguanosine; arachaeosine [also called 7-formamido-7-deazaguanosine]; and 7-aminomethyl-7-deazaguanosine.

Another modification of the antisense oligonucleotides of the disclosure involves chemically linking to the nucleic acid sequences one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al. Ann. N.Y. Acad. Sci. 1992, 660, 306; Manoharan et al. Bioorg. Med. Chem. Let. 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al. EMBO J. 1991, 10, 111; Kabanov et al. FEBS Lett. 1990, 259, 327; Svinarchuk et al. Biochimie 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651; Shea et al. Nucl. Acids Res. 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al. Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651). It is not necessary for all positions in a given nucleic acid sequence to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single nucleic acid sequence or even at within a single nucleoside within a nucleic acid sequence.

Vectors

A vector is a macromolecule or association of macromolecules that comprises or associates with one or more polynucleotides (or an expression vector comprising such polynucleotide(s)) and which can be used to mediate delivery of the polynucleotide(s) to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. A vector can be combined with a lipid, polymer carrier, or any other suitable carrier. The vector may comprise regulatory elements not provided by the expression vector, which become operatively linked to the polynucleotide(s) when they or the expression vector comprising them is inserted into the vector. A vector can be engineered to lack one or more elements for vector replication.

In some embodiments, a vector can comprise the nucleic acid expression cassettes described herein. In some embodiments, the vector can be a viral vector or a plasmid vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector or plasmid vector complexed with lipid or polymer carrier.

Viral gene therapy vectors or gene delivery vectors can have the ability to be reproducibly and/or stably propagated and purified to high titers; to mediate targeted delivery (e.g., to deliver the polynucleotide specifically to a tissue or organ of interest without widespread vector dissemination elsewhere or off-target delivery); and to mediate gene delivery and/or polynucleotide expression without inducing harmful side effects or off-target effects.

The term “AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or a derivative thereof. The term covers all serotypes, subtypes, and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”). The term “AAV” includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. An “rAAV vector” as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs). The term “rAAV vector” encompasses both rAAV vector particles and rAAV vector plasmids. An rAAV vector may either be single-stranded (ssAAV) or self-complementary (scAAV). An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a polynucleotide or a nucleic acid expression cassette to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector.” Thus, production of rAAV particle necessarily includes production of an rAAV vector, as such a vector is contained within an rAAV particle.

The cloning capacity of vectors or viral expression vectors can be a particular challenge for expression of large polynucleotides. For example, AAV vectors typically have a packaging capacity of ˜4.8 kb, lentiviruses typically have a capacity of ˜8 kb, adenoviruses typically have a capacity of ˜7.5 kb, and alphaviruses typically have a capacity of −7.5 kb. Some viruses can have larger packaging capacities, for example herpesvirus can have a capacity of >30 kb and vaccinia a capacity of ˜25 kb. Advantages of using AAV for gene therapy include low pathogenicity, very low frequency of integration into the host genome, and the ability to infect dividing and non-dividing cells.

Several serotypes of AAV, non-pathogenic parvovirus, have been engineered for the purposes of gene delivery, some of which are known to have tropism for certain tissues or cell types. Viruses used for various gene-therapy applications can be engineered to be replication-deficient or to have low toxicity and low pathogenicity in a subject or a host. Such virus-based vectors can be obtained by deleting all, or some, of the coding regions from the viral genome, and leaving intact those sequences (e.g., inverted terminal repeat sequences) that are necessary for functions such as packaging the vector genome into the virus capsid or the integration of vector nucleic acid (e.g., DNA) into the host chromatin. A nucleic acid expression cassette comprising a polynucleotide, for example, can be cloned into a viral backbone such as a modified or engineered viral backbone lacking viral genes, and used in conjunction with additional vectors (e.g., packaging vectors), which can, for example, when co-transfected, produce recombinant viral vector particles.

In some cases, an AAV vector or an AAV viral particle, or virion, used to deliver a nucleic acid expression cassette into a cell, cell type, or tissue, in vivo or in vitro, is replication-deficient. In some cases, an AAV virus is engineered or genetically modified so that it can replicate and generate virions only in the presence of helper factors.

In some embodiments, a nucleic acid expression cassette is designed for delivery by an AAV or a recombinant AAV (rAAV). In some embodiments, a nucleic acid expression cassette is delivered using a lentivirus or a lentiviral vector. In some embodiments, larger polynucleotide, i.e., genes that exceed the cloning capacity of AAV, are preferably delivered using a lentivirus or a lentiviral vector.

In some embodiments, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVDJ, AAVrh10, AAV11, AAV12, AAV2/1, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/11, or AAV2/12, AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49, AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/r11.64, AAV4-9/rh.54, AAV4-19/rh.55, AAV5-3/rh.57, AAVS-22/rh.58, AAV7.3/hu.7, AAV16.8/hu.10, AAV16.12/hu.11, AAV29.3/bb.1, AAV29.5/bb.2, AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R AS86R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, and/or AAVF9/HSC9 and variants thereof. PHP.B, and PHP.B derivatives [PHP.eR, PHP.S], AAV8[K137R] AAV-TT, rAAV-retro, AAV9.HR, AAV1 CAM mutants, AAV9[586-590] swap mutants. In some embodiments, the AAV vector is a hybrid or chimeric AAV serotype. In some embodiments, the AAV is an engineered AAV designed to modify tropism or evade immune detection.

In some embodiments, the nucleic acid expression cassette can be designed for delivery by an optimized therapeutic retroviral vector, e.g., a lentiviral vector. The retroviral vector can be a lentiviral vector comprising a left (5′) LTR; sequences which aid packaging and/or nuclear import of the virus, at least one regulatory element, optionally a lentiviral Rev response element (RRE); optionally a promoter or active portion thereof; a polynucleotide operably linked to one or more regulatory elements; optionally an insulator; and a right (3′) retroviral LTR. A lentiviral vector can also include a posttranscriptional regulatory element, such as the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and/or any of the transcriptional and posttranscriptional regulatory elements described herein. A lentiviral vector can be a self-inactivating (SIN) lentiviral vector. Any suitable packaging system can be used with a lentiviral vector, including second, third, and fourth generation packaging systems, for example. A lentiviral vector can be pseudotyped. Any envelope glycoprotein can be used for pseudotyping, including, for example, a glycoprotein from vesicular stomatitis virus (VSV), rabies virus, Lyssavirus, Mokola virus, lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), retroviruses, Moloney murine leukemia virus (MuLV), filoviruses, paramyxoviruses, measles virus, Nipah virus, orthomyxoviruses, and others. A lentiviral vector can be pseudotyped to alter tropism. Any cell type can be targeted by pseudotyping, including neuronal cells, for example.

Also provided herein are vectors or sets of vectors comprising: (i) a vector comprising an expression cassette provided herein; or (ii) a set of vectors comprising (a) a first vector comprising a first polynucleotide provided herein (e.g., an antisense oligonucleotide coding sequence), and (b) a second vector comprising a second polynucleotide provided herein (e.g., a wild-type PSEN1 or PSEN2 coding sequence resistant to silencing by the encoded antisense oligonucleotide.

Techniques contemplated herein for gene therapy of somatic cells include delivery via a viral vector (e.g., retroviral, adenoviral, AAV, helper-dependent adenoviral systems, hybrid adenoviral systems, herpes simplex, pox virus, lentivirus, and Epstein-Barr virus), and non-viral systems, such as physical systems (naked DNA, DNA bombardment, electroporation, hydrodynamic, ultrasound, and magnetofection), and chemical systems (cationic lipids, different cationic polymers, and lipid polymers).

Delivery Routes

In certain embodiments, the expression cassettes and vectors disclosed herein can be formulated in any suitable formulation suitable for a particular route of administration. Various pharmaceutically acceptable formulations are commercially available and obtainable by a medical practitioner.

In certain embodiments, the expression cassettes and vectors disclosed herein are administered to the central nervous system (CNS) of a subject in need. In certain embodiments, the central nervous system includes brain, spinal cord and cerebral spinal fluid (CSF). In certain embodiments, the compositions are administered to the brain or spinal cord or CSF of a mammal. In certain embodiments, the compositions are administered to a portion of brain or spinal cord.

In certain embodiments, the expression cassettes and vectors disclosed herein are administered to brain parenchyma, subarachnoid space and/or intrathecal space. In certain embodiments, the compositions are administered to one or more of cisterna magna, intraventricular space, brain ventricle, subarachnoid space, and/or ependyma of said subject.

In further embodiments, the expression cassettes and vectors disclosed herein are administered to the ventricular system. In still further embodiments, the expression cassettes and vectors disclosed herein are administered to one or more of the rostral lateral ventricle; and/or caudal lateral ventricle; and/or right lateral ventricle; and/or left lateral ventricle; and/or right rostral lateral ventricle; and/or left rostral lateral ventricle; and/or right caudal lateral ventricle; and/or left caudal lateral ventricle.

In certain embodiments, the expression cassettes and vectors disclosed herein are administered to one or more cells that contact the CSF in a mammal, for example by contacting cells with the compositions. Non-limiting examples of cells that contact the CSF include ependymal cells, pial cells, endothelial cells and/or meningeal cells. In certain embodiments, the expression cassettes and vectors disclosed herein are administered to ependymal cells. In certain embodiments, the expression cassettes and vectors disclosed herein are delivered to ependymal cells, for example by contacting ependymal cells with the compositions.

In certain embodiments, the expression cassettes and vectors disclosed herein are administered/delivered locally. “Local delivery” refers to delivery directly to a target site within a mammal (e.g., directly to a tissue or fluid). For example, the expression cassettes and vectors disclosed herein can be locally delivered by direct injection into an organ, tissue or specified anatomical location. In certain embodiments, the expression cassettes and vectors disclosed herein are delivered or administered by direct injection to the brain, spinal cord, or a tissue or fluid thereof (e.g., CSF, such as ependymal cells, pial cells, endothelial cells and/or meningeal cells). For example, the expression cassettes and vectors disclosed herein can be directly delivered, by way of direct injection, to the CSF, cisterna magna, intraventricular space, a brain ventricle, subarachnoid space and/or intrathecal space; and/or ependymal; and/or rostral lateral ventricle; and/or caudal lateral ventricle; and/or right lateral ventricle; and/or left lateral ventricle; and/or right rostral lateral ventricle; and/or left rostral lateral ventricle; and/or right caudal lateral ventricle; and/or left caudal lateral ventricle.

In certain embodiments, the expression cassettes and vectors disclosed herein are delivered to a tissue, fluid or cell of the brain or spinal cord by direct injection into a tissue or fluid of the brain or spinal cord. In certain embodiments, the expression cassettes and vectors disclosed herein are not delivered systemically by, for example, intravenous, subcutaneous, or intramuscular injection, or by intravenous infusion. In certain embodiments, the expression cassettes and vectors disclosed herein are delivered to a tissue or fluid of the brain or spinal cord by stereotactic injection.

In certain embodiments, the expression cassettes and vectors disclosed herein are delivered or administered by direct injection to the brain, spinal cord, or portion thereof, or a tissue or fluid thereof (e.g., CSF such as ependyma).

In certain embodiments, a method or use includes administering the expression cassettes and vectors disclosed herein to the brain or spinal cord, or portion thereof, of a human. In certain embodiments, the wild-type PSEN1 or PSEN2 polypeptides (and the antisense oligonucleotides when encoded by an expression vector) are expressed and/or detected in a central nervous tissue (e.g., brain, e.g., striatum, thalamus, medulla, cerebellum, occipital cortex, prefrontal cortex) distal to the administration site. In certain embodiments, the polypeptide is present or detected broadly in a central nervous tissue (e.g., brain, e.g., striatum, thalamus, medulla, cerebellum, occipital cortex, and/or prefrontal cortex) that reflects distribution away from the administration site and optionally throughout a central nervous tissue (e.g., brain, e.g., striatum, thalamus, medulla, cerebellum, occipital cortex, and/or prefrontal cortex).

An effective amount of the expression cassettes and vectors disclosed herein, such as rAAV vector expressing the PSEN1 or PSEN2, an antisense oligonucleotide or both, can be empirically determined. Administration can be effected in one or more doses, continuously or intermittently throughout the course of treatment. Effective doses of administration can be determined by those of skill in the art and may vary according to the AAV serotype, viral titer and the weight, condition and species of mammal being treated. Single and multiple administrations (e.g., 1-5 or more) can be carried out with the dose level, target and timing being selected by the treating physician. Multiple doses may be administered as is required to maintain adequate enzyme activity, for example.

The expression cassettes and vectors disclosed herein, can be administered as a part of a combination therapy, for example, a subject with dementia or Alzheimer's Disease, with one or more additional therapeutic agents. For example, the U.S. Food and Drug Administration (FDA) has approved two types of medications—cholinesterase inhibitors (ARICEPT®, EXELON®, RAZADYNE®) and memantine (NAMENDA®)—to treat the cognitive symptoms (memory loss, confusion, and problems with thinking and reasoning) of Alzheimer's disease. For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of the other agent.

When “co-administered” with other agents, e.g., when co-administered with another medication, an “effective amount” of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a compound described herein being used.

Kits

Provided herein are kits comprising one or more vectors or sets of vectors described herein. In some embodiments, the kit comprises: a) one or more antisense oligonucleotides, wherein each antisense oligonucleotide independently targets either a coding region or a non-coding region of an mRNA translated from each of a human wild-type and mutant presenilin 1 (PSEN1), each of a human wild-type or mutant presenilin 2 (PSEN2); and b) a vector comprising a polynucleotide encoding a wild-type presenilin 1 (PSEN1) amino acid sequence or a wild-type presenilin 2 (PSEN2) amino acid sequence, wherein the second polynucleotide is not targeted by any of the one or more antisense oligonucleotides; and wherein the polynucleotide is operably linked to a promoter in the vector. In some aspects of these embodiments, each of the one or more antisense oligonucleotides is independently selected from a short hairpin RNA (shRNA), a short interfering RNA (siRNA), a micro interfering RNA (miRNA), a small temporal RNA (stRNA) or an endoribonuclease-prepared siRNA (esiRNA). In some aspects of these embodiments, at least one of the one or more antisense oligonucleotides in the kit comprises one or more modified nucleobases. In some more specific aspects of these embodiments, each of the one or more modified nucleobases is independently selected from a non-naturally occurring nucleobase, a locked nucleic acids (LNA), or a peptide nucleic acids (PNA).

In addition to the active components (e.g., vectors and/or antisense oligonucleotides), the kits of the present disclosure may comprise one or more of any of the following: instructions for preparing the active components for administration to a subject, instructions for administering the active components to a subject, buffers, diluents, solvents, or other excipients to dissolve and/or dilute and/or prepare any of the active components for administration to a subject, extra vessels for diluting or dividing the active components, tools for administering the active components, and any other items that are useful in using the active components in therapy.

Methods of Treatment

The present polynucleotide sequences, antisense oligonucleotides, expression cassettes, vectors, sets of vectors, and kits are useful in methods of treating any disorder characterized by a mutant form of PSEN1 or PSEN2. Such methods comprise the step(s) of administering an antisense oligonucleotide (or a polynucleotide that encodes such antisense oligonucleotide) that targets PSEN1 or PSEN2; and a polynucleotide that encodes wild-type PSEN1 or PSEN2 and which is resistant to silencing by the antisense oligonucleotide. In some embodiments, these two components may be encoded in a single expression cassette or vector administered to a subject. In some embodiments, these two components may be encoded in separate expression cassettes or vectors administered sequentially in any order, or simultaneously to a subject. In some embodiments, the antisense oligonucleotide may be administered directly to the subject sequentially in any order, or simultaneously with a vector or expression cassette encoding the wild-type PSEN1 or PSEN2 protein.

Diseases and disorders useful in these methods include any neurodegenerative disease, disorder, or condition characterized by a mutant form of PSEN1 or PSEN2. In some embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease, familial Alzheimer's disease, sporadic Alzheimer's disease, late-onset Alzheimer's disease, frontotemporal dementia, frontotemporal lobar degeneration, Pick's disease, Lewy body dementia, memory loss, cognitive impairment, or mild cognitive impairment. Other exemplary neurodegenerative diseases, disorders, or conditions include tauopathy, primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), amyotrophic lateral sclerosis-parkinsonism-dementia (ALS-PDC, Lytico-bodig disease), ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, synucleinopathy, Parkinson's disease, multiple system atrophy (MSA), neuraxonal dystrophies, Parkinson's-like disease, Parkinsonism, prion diseases, motor neuron diseases, dementia, transmissible spongiform encephalopathies, systemic atrophies primarily affecting the central nervous system, trinucleotide repeat disorders, proteopathies, amyloidosis, neuronal ceroid lipofuscinoses, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, seizure disorders, paraplegias, demyelinating diseases, Huntington's disease, traumatic brain injury, stroke, autism spectrum disorder (ASD), depression, anxiety, post-traumatic stress disorder (PTSD), schizophrenia, Attention-Deficit/Hyperactivity Disorder (ADHD), bipolar disorder, Obsessive-Compulsive Disorder (OCD), personality disorder, pain, and others.

As used herein, the terms “treat,” “treatment,” “therapy,” “therapeutic,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect, including, but not limited to, alleviating, delaying or slowing the progression, reducing the effects or symptoms, preventing onset, inhibiting, ameliorating the onset of a diseases or disorder, obtaining a beneficial or desired result with respect to a disease, disorder, or medical condition, such as a therapeutic benefit and/or a prophylactic benefit. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease. In some embodiments, the methods disclosed herein are useful to preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it, or in a subject that possesses biomarkers associated with the disease but does not yet show any physical symptoms of the disease.

A therapeutic benefit includes eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. In some cases, for prophylactic benefit, the compositions are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. The methods of the present disclosure may be used with any mammal or other animal. In some cases, the treatment can result in a decrease or cessation of symptoms. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

A subject is any individual or patient on which the methods disclosed herein are performed. The term “subject” can be used interchangeably with the term “individual” or “patient.” The subject can be a human, although the subject may be an animal, as will be appreciated by those in the art. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject. In some embodiments, the subject is a human.

Expression cassettes and vectors provided herein can be administered in an amount effective to treat the neurodegenerative disease, disorder, or condition, The term “effective amount” or “therapeutically effective amount” refers to that amount of a composition described herein that is sufficient to affect the intended application, including but not limited to disease treatment, as defined herein. The therapeutically effective amount may vary depending upon the intended treatment application (in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in a target cell. The specific dose will vary depending on the particular composition chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

Expression cassettes and vectors can be delivered by any suitable method. Exemplary methods include intracranial injection, stereotaxic injection into the brain grey or white matter, injection into the cerebrospinal fluid (intrathecal, intracerebroventricular, intracisternal-magna), and intravenous injection.

The procedures described herein employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. (See, e.g., Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1982); Sambrook, et al., (1989); Sambrook and Russell, Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons (including periodic updates) (1992); Glover, DNA Cloning, IRL Press, Oxford (1985); Russell, Molecular biology of plants: a laboratory course manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); Anand, Techniques for the Analysis of Complex Genomes, Academic Press, NY (1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology, Academic Press, NY (1991); Harlow and Lane, Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988); Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds. (1984); Culture Of Animal Cells, R. I. Freshney, A. R. Liss, Inc. (1987); Immobilized Cells And Enzymes, IRL Press (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology, Academic Press, Inc., NY); Methods In Enzymology, Vols. 154 and 155, Wu, et al., eds.; Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds. (1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford (1988); Fire, et al., RNA Interference Technology From Basic Science to Drug Development, Cambridge University Press, Cambridge (2005); Schepers, RNA Interference in Practice, Wiley-VCH (2005); Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press (2003); Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, N.J. (2004); and Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC (2004)).

The compositions and methods are more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. The term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105).

The terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined below to provide additional guidance to the practitioner regarding the description of the compositions and methods.

All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements or steps that are included in the description herein can be excluded from the claimed compositions or methods

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

The following are provided for exemplification purposes only and are not intended to limit the scope of the invention described in broad terms above.

EXAMPLE 1
Design of siRNA Sequences for Silencing Endogenous PSEN1 Gene Expression in Humans

We designed siRNA sequences that can target endogenous human PSEN1 mRNAs. These siRNA sequences may be used for direct administration to a subject or encoded by a polynucleotide as part of a shRNA or miRNA that is produced from a vector administered to the subject.

Once these siRNAs are used to inhibit endogenous PSEN1 genes, PSEN 1 expression can be restored by providing a PSEN1 cDNA for expression of an mRNA encoding a wild-type presenilin 1 protein and resistant to suppression by such siRNAs by codon modification or otherwise excluding the shRNA target sequences from the mRNA.

siRNA sequences were designed using known art and principles in molecular biology including the use of online tools, including siRNA designer at Integrated DNA Technologies (IDT; biotools.idtdna.com/site/order/designtool/index/DSIRNA_CUSTOM), siDirect (sidirect2.rnai.jp/), and Thermo Fisher (https://rnaidesigner.thermofisher.com/rnaiexpress/).

A set of potential targets for siRNA were identified in either the protein encoding region or the non-coding region of the human PSEN1 mRNA. The DNA sequences encoding the corresponding siRNA sequences that target PSEN1 mRNA are set forth in SEQ ID NOS: 1-32, 42 and 43. Table 2, below, shows the PSEN1 target location in the GenBank NM 000021.4 PSEN 1 cDNA sequence (and therefore the corresponding location in the transcribed PSEN1 mRNA) to which the encoded siRNA would hybridize. Sequences within a complementary location in NM_000021.4 between 213-1616 are within the PSEN1 protein-encoding region.

TABLE 2

DNA sequences encoding siRNA that target endogenous

PSEN1 mRNA and complementary location of targeting within

the GenBank NM_000021.4 cDNA sequence.

Complementary Location

SEQ ID NO:
in NM_000021.4

1
94-115

2
176-201

3
402-424

4
452-472

5
607-632

6
621-643

7
630-655

42
953-973

8
1026-1048

9
1091-1113

10
1285-1307

11
1410-1433

43
1581-1601

12
1630-1654

13
1638-1660

14
1697-1722

15
1930-1954

16
1916-1940

17
2950-2975

18
4522-4547

19
5931-5956

A similar list was generated for DNA sequences that encode PSEN2-specific siRNA (SEQ ID NOS: 20-32). Table 3, below, shows the PSEN2 target location in the GenBank NM_000447.3 PSEN2 cDNA sequence (and therefore the corresponding location in the transcribed PSEN2 mRNA) to which the encoded siRNA would hybridize. Sequences with a complementary location in NM_000447.3 between 384-1730 are within the PSEN2 mRNA coding region.

TABLE 3

DNA sequences encoding siRNA that target endogenous

PSEN2 mRNA and complementary location of targeting within

the (Genbank NM_000447.3) cDNA sequence.

Complementary Location

SEQ ID NO:
in NM_000447.3

20
110-135

21
143-168

22
735-760

23
736-761

24
737-762

25
819-844

26
849-874

27
1349-1374

28
1766-1791

29
1771-1796

30
1773-1798

31
1962-1987

32
2201-2226

These PSEN1- and PSEN2-specific siRNA encoding sequences, or other DNA sequences encoding a siRNA that hybridizes to endogenous PSEN2 mRNA and which comprise at least 7 or more consecutive nucleotides from either the 5′ or 3′ end of such sequences, can be used in a polynucleotide encoding an shRNA or an miRNA that targets endogenous PSEN1 or PSEN2 mRNA.

We next designed exogenous DNA molecules that, when introduced to target cells and transcribed into RNA, silence the translation of endogenous PSEN1 or PSEN2 mRNA, including both mRNA transcribed from wild-type alleles and mutant alleles, if present. These DNA molecules included shRNA-encoding molecules (SEQ ID NO:44-47) and miRNA-encoding molecules (SEQ ID NO: 33-36). See Table 4, below, which shows the type of antisense oligonucleotide encoded, the PSEN targeted and the target location in the corresponding GenBank cDNA sequence (and therefore the corresponding location in the transcribed PSEN mRNA) to which the siRNA would hybridize.

TABLE 4

miRNA sequences. The underlined siRNA coding sequences

are used to generate artificial miRNAs or Pre-miRNAs

Complementary Target

PSEN
Location in Corresponding

SEQ ID NO:
Type
Targeted
GenBank cDNA Sequence

33
miRNA
PSEN1
1631-1652

34
miRNA
PSEN2
1766-1788

35
miRNA
PSEN1
1631-1651

36
miRNA
PSEN2
1768-1790

44
shRNA
PSEN1
401-421

45
shRNA
PSEN1
953-973

46
shRNA
PSEN1
1285-1305

47
shRNA
PSEN1
1581-1601

In addition to these, other complementary locations targeted in GenBank NM_000021.4 and NM_000447.3 cDNA sequences are represented by the siRNA sequences embedded in miRNA targeting sequences and encoded by the plasmids set forth in SEQ ID NOs: 68-81. These are set forth below in Table 4A

TABLE 4A

Additional miRNA sequences and complementary targets

Complementary

Nucleotides encoding

Target Location

miRNA Targeting
Nucleotides

in Corresponding

Source
Sequence(s) (including
encoding siRNA
PSEN
GenBank cDNA

SEQ ID NO:
siRNA sequence(s))
Sequence(s)
Targeted
Sequence

68
448-529
497-517
PSEN1
1639-1659

69
448-529
497-517
PSEN1
1697-1717

70
448-529
497-517
PSEN1
1796-1816

71
448-529
497-517
PSEN1
1930-1950

72
448-611¹
497-517,
PSEN1
1796-1816

579-599

73
448-693²
497-517,
PSEN1
1639-1659

579-599,

661-681

74
448-693²
497-517,
PSEN1
1796-1816

579-599,

661-681

75
1365-1610²
1414-1434,
PSEN1
1796-1816

1496-1516,

1578-1598

76
448-529
497-517
PSEN2
1771-1791

77
448-529
497-517
PSEN2
1778-1798

78
448-529
497-517
PSEN2
2206-2226

79
448-611¹
497-517,
PSEN2
2206-2226

579-599

80
448-693²
497-517,
PSEN2
2206-2226

579-599,

661-681

81
1365-1610²
1414-1434,
PSEN2
2206-2226

1496-1516,

1578-1598

¹comprises two consecutive copies of the miRNA.

²comprises three consecutive copies of the miRNA.

Any of the polynucleotide sequences encoding shRNAs or miRNAs can be delivered simultaneously or consecutively with a polynucleotide that also expresses an mRNA encoding wild-type PSEN1 or PSEN2 that is resistant to silencing by the co-delivered shRNA or miRNA. The DNA encoding PSEN1 or PSEN2 mRNA and silencer polynucleotides may be delivered as polynucleotides in a single DNA vector or as a replication-deficient adeno-associated virus (AAV) vector. Alternatively, the polynucleotide encoding the shRNA or miRNA may be delivered in a separate DNA vector or AAV vector from the polynucleotide encoding PSEN1 or PSEN2 mRNA.

An encoded shRNA comprises between about 20-25 nucleotides that are identical to a portion of the target mRNA sequence followed by a linker and a sequence complementary to that same portion of the target mRNA. shRNAs are expressed from the DNA encoding them, which is typically operably linked to an RNA polymerase III driven promoter, such as U6, U61, U69, or H1. From one to four shRNAs, each targeting a different portion of the endogenous PSEN1 or PSEN2 mRNA are expressed from the same DNA or AAV vector to mediate degradation of the endogenous PSEN1 or PSEN2 mRNA and reduce PSEN1 or PSEN2 protein levels.

Some of the PSEN1 targets (the portions PSEN1 mRNA targeted by SEQ ID NOs:6, 11, and 42) are also present in the mouse PSEN1 mRNA (see the corresponding mouse PSEN1 cDNA sequence GenBank NM_001362271.1). Therefore, antisense oligonucleotides targeting those sequences will also suppress expression of endogenous mouse PSEN1 gene. These antisense oligonucleotides may be used as a tool for in vivo assessment in mouse models of Alzheimer's disease of the efficacy of antisense molecules and vectors suppressing endogenous PSEN1 gene with simultaneous replacement by a PSEN1 gene resistant to suppression.

In the dominant forms of PSEN1 deficiency, the expression of a mutant PSEN1 subunit inhibits the assembly and function of the gamma secretase. Without being limited by theory, we believe that a simple gene replacement method would provide more wildtype PSEN1 subunit of gamma secretase, but not suppress the inhibitory effect of the mutant subunit on assembly and/or function. However, by suppressing all endogenous PSEN1 expression and replacing it with an extra-chromosomally expressed wild-type PSEN1, the full gamma secretase activity can be restored. In addition to treating Alzheimer's disease or ameliorating the increased susceptibility to Alzheimer's disease that results from dominant mutations of the PSEN1 gene, the concept of silencing endogenous PSEN1 (or PSEN2) gene expression and replacement with a gene encoding a wild-type form of that protein and resistant to silencing can be applied to any disease which involves a defect in PSEN1 (or PSEN2).

EXAMPLE 2
Codon Changes to Escape Silencing by shRNA Targeting Native PSEN1 mRNA

We designed replacement PSEN1 genes that encode the native PSEN1 protein sequence, but whose encoded mRNA is not recognized by the shRNA targeting the endogenous PSEN1 by one of two methods:

1) If the shRNA(s) are designed to exclusively target PSEN1 mRNA outside the protein coding portion, then the replacement PSEN1 coding sequence can be identical to the portion of the endogenous mRNA that encodes the wild-type protein. The expression vector used to express the replacement PSEN 1 coding sequence encodes both upstream and downstream non-coding portions of mRNA from a totally unrelated source that will not hybridize to the shRNA(s).
2) If any of the shRNA(s) target endogenous PSEN1 mRNA within protein encoding regions, the replacement PSEN1 coding sequence is codon-modified and uses synonymous codons that provide the same amino acids sequence. By utilizing such modified codons, the ability of the shRNA(s) to target the mRNA expressed from replacement PSEN1 coding sequence is eliminated or greatly reduced. The ability to change synonymous codons depends on the amino acid sequence encoded by the target sequence within the mRNA. Ideally, a sufficient number of codons are changed in the shRNA targeted sequence to provide at least a 50%, at least 40%, at least 30% or at least 20% nucleotide differences, or at least 4 or at least 5 mismatches from the antisense portion of the shRNA.

The codon modifications only needs to occur in those portions of the coding sequence targeted by shRNA. Therefore, the replacement PSEN1 coding sequence can be identical to the endogenous nucleotide sequence throughout most of the coding region, with only a few regions of codon modification.

The same procedure is used to generate the codon modified PSEN2 nucleic acid sequence.

EXAMPLE 3
In Vitro siRNA Suppression of PSEN1

Bioinformatic assessment is used to select in silico highly specific siRNA sequences and minimize cross-reactivity. Oligonucleotides complementary to PSEN1 are designed and synthesized for specifically binding to PSEN1 and degradation of PSEN1 mRNA by the RNA interference pathway.

PSEN1 suppression is evaluated in commercial cell lines like HEK293 or Hela cells by transfection or direct incubation of the oligonucleotides. When cells reached 65-75% confluency, the transfection reagent, for example LIPOFECTIN is used to introduce the oligonucleotide into cells. Other methods of transfection are well known to those skilled in the art. The method of screening is not a limitation of the instant invention. The oligonucleotide is mixed with LIPOFECTIN (Invitrogen Life Technologies) in culture media like, OPTI-MEM-1 (Invitrogen Life Technologies) to achieve the desired concentration of oligonucleotide and a LIPOFECTIN concentration. Cells are treated and data are obtained in duplicate or triplicate. After treatment, the medium containing the transfection mixture is replaced with fresh culture medium. Cells are harvested 16-24 hours after oligonucleotide treatment.

Quantitation of PSEN1 mRNA levels is accomplished by real-time quantitative PCR. After isolation from cells, RNA is subjected to sequential reverse transcriptase (RT) reaction and real-time PCR. RT and PCR reagents can be obtained from Invitrogen Life Technologies. RT, real-time PCR is carried out according to manufacturer's instructions using primers and probe set specific for PSEN1 and the real-time PCR data is normalized to a house keeping gene whose expression is constant. The percent of inhibition of PSEN1 mRNA levels relative to control scrambled or untreated cells is calculated. The target regions to which antisense oligonucleotides are inhibitory are used to design shRNA and miRNA.

EXAMPLE 4
AAV Vector with a PSEN1 Silence and Replace System to Suppress Expression of Mutant PSEN1 and Express Wild-Type PSEN1

An adenovirus associated virus (AAV) vector is constructed to contain miRNA which target and cleave PSEN1 mRNA and the coding sequence for wild-type PSEN1. The AAV viral vector containing a genome construct which encodes both the miRNA and coding sequence is derived from a commercially available plasmid-based expression vector. The commercial plasmid is modified to include inverted terminal repeats (ITR) of an AAV2, U6 a polymerase III promoter, three miRNA sequences targeting the PSEN1 gene with binding sites in the 3′UTR, CBA a polymerase II promoter, the coding sequence for wild-type PSEN1 followed by a rabbit beta-globin polyadenylation sequence and another AAV2 ITR (SEQ ID NO:37,38).

Production of AAV viral particles with PSEN1 silence and replace genome is accomplished by co-transfection of human embryonic kidney (HEK293) or insect (Sf9) cells with the AAV viral vector genome plasmid and helper plasmids to supply protein essential to AAV and a plasmid to express viral capsid proteins. Methods and cell lines for producing AAV particles are well known to those skilled in the art. Following culture, the viral particles are harvested and concentrated to achieve viral genome copy numbers in range between 10¹¹-10¹³VG/mL (see e.g., Chen et al, Human Gene Therapy Methods 24: 270-278, 2013).

EXAMPLE 5
In Vitro and In Vivo Testing of AAV Vector with PSEN2 Silence and Replace

An adenovirus associated virus (AAV) vector is constructed to contain elements of a contain miRNA which target and cleave PSEN2 mRNA and the coding sequence for wild-type PSEN2. The elements of a PSEN2 silence and replace system are delivered by an AAV vector which efficiently transduces mammalian tissues and resides long term in the cell nucleus as an epichromosome.

AAV particles containing PSEN2 silence and replace system may be tested in vitro using a mammalian cell line, e.g. HEK293 cells (available from American Type Culture Collection, Manassas, Va.). Transduction of mammalian cells with AAV vectors in vitro is described (see e.g., Le Cong et al, Ibid., and Sen et al, Scientific Reports 3: 1832, 2013; DOI: 10.1038/srep01832 which is incorporated herein by reference). Following transduction, the endogenous and exogenous PSEN2 transcripts may be monitored by quantitative RT-PCR (qRT-PCR) using established methods (see e.g., Perez-Pinera et al, Nature Methods Advance Online Publication, July 25, 2013; doi: 10.1038/nmeth.2600 which is incorporated herein by reference).

In order to evaluate the effects of containing PSEN2 silence and replace system in the central nervous system, it is beneficial to deliver the AAV vector directly to the central nervous system, for example by intracerebroventricular (ICV) or intracisternal magna (ICM) administrations. To evaluate the effect of PSEN2 silence and replace system in the central nervous system of animals, AAV can be administered to mice via ICV delivery.

Selected AAV vectors containing potent shRNAs or miRNAs and encoding PSEN2 can be used for in vivo testing. Formulation-treated mice can be used as control animals. Each treatment or control groups may include 4-12 animals. AAV is administered ICV at a dose of 10¹⁰-10¹¹viral genomes. The treatment period may be four-weeks. During the treatment period, the mice are monitored for clinical changes such as body weight changes or abnormal behaviors. At the end of the treatment period, the mice are sacrificed, and the brain is dissected. RNA is prepared for quantitative real-time PCR analysis and brain homogenates are used for PSEN2 protein quantification by ELISA and characterization by western blot.

EXAMPLE 6
In Vivo PSEN1 Silence and Replace in Models of Alzheimer's Disease

To evaluate the effects of PSEN1 silence and replace in the central nervous system of an animal model of AD, AAV encoding PSEN1 silence and replace are administered to PSEN1 knockin (KI) mice carrying the FAD mutation L435F. KI mice heterogenous for either mutation in a background lacking PSEN2, Psen1^L435F/+; Psen2^−/− (Xia et al., Neuron. 2015 doi: 10.1016/j.neuron.2015.02.010). The L435F mutation abolished the production of mature PSEN1 (N-terminal and C-terminal fragments) without any change in PSEN1 mRNA levels. The Psen1^L435F/+; Psen2^−/− transgenic mouse model shows accelerated amyloid deposition, impaired hippocampal synaptic plasticity and memory, and cerebral cortical neurodegeneration reminiscent of AD.

To evaluate the effects of PSEN1 silence and replace in animal model of AD, AAV encoding PSEN1 silence and replace system is administered to Psen1^L435F/+; Psen2^−/− transgenic mouse model via ICV delivery. Selected AAV vectors containing potent shRNAs or miRNAs and encoding PSEN1 can be used for in vivo testing. Formulation-treated mice can be used as control animals. Each treatment or control groups may include 4-12 animals. AAV is administered ICV at a dose of 10¹⁰-10¹¹viral genomes. The treatment period may be six to eighteen months. During the treatment period, the mice are monitored for clinical changes such as body weight changes or abnormal behaviors. At the end of the treatment period, the mice are sacrificed, and the brain is dissected. RNA is prepared for quantitative real-time PCR analysis and brain homogenates are used for PSEN1 protein quantification by ELISA and characterization by western blot.

EXAMPLE 7
PSEN1 and PSEN2 Silence and Replace Plasmids

A plasmid comprising AAV2 ITRs (nucleotides 1-141 and 4298-4438 of SEQ ID NO:68), a U6 promoter (nucleotides 198-241 of SEQ ID NO:68), a CMV enhancer (nucleotides 561-940 of SEQ ID NO:68), a CBA promoter (nucleotides 941-1213 of SEQ ID NO:68), an HA epitope tag (nucleotides 1873-1905 of SEQ ID NO:68), a codon optimized human PSEN1 coding sequence (“hPSEN1v1.5”; nucleotides 1906-3303 of SEQ ID NO:68) or a human PSEN2 coding sequence (nucleotides 1902-3245 of SEQ ID NO:76) functionally linked to that CBA promoter, and a human growth hormone (hGH) PolyA signal (nucleotides 3337-3813 of SEQ ID NO: 68) was used as the backbone to generate the human silence and replace constructs. Into those plasmids at various sites was inserted one, two or three copies of a nucleotide sequence consisting of a miR128 targeting sequence flanking an siRNA sequence that was complementary to a different region of either native PSEN1 or native PSEN2. The resulting plasmids (SEQ ID NOs: 68-81; FIGS. 1-14) were separately transfected into HEK293 (ATCC° CRL-1573TM) cells using standard techniques. The HEK293 cells were harvested 48 hours post-transfection, lysed directly using 500 μL of QIAzol Lysis Reagent (Qiagen, #79306), and the supernatant was collected. Following lysis, the samples were homogenized using a Qiashredder (Qiagen, #79656) and RNA was isolated using RNeasy Plus Universal Mini kit (Qiagen, #74034) according to the manufacturers protocol. The total RNA concentration of each sample was measured using the DeNovix DS-11 FX+ spectrophotometer/fluorometer according to the manufacturer's instructions. After isolation from cells, RNA was subjected to sequential reverse transcriptase (RT) reaction and real-time PCR. RT and PCR reagents were obtained from ThermoFisher Scientific. RT and real-time PCR was carried out according to manufacturer's instructions using primers and probe set specific for (a) native PSEN1 that did not recognize the codon-optimized human PSEN1 present on the codon-optimized PSEN1-encoding plasmids (forward primer =SEQ ID NO:82; probe=SEQ ID NO: 83; reverse primer=SEQ ID NO:84); (b) plasmid-encoded transcripts (specific for the hGHpolyA stuffer present in the plasmids; forward primer=SEQ ID NO:85; probe=SEQ ID NO: 86; reverse primer=SEQ ID NO:87), or (c) a non-coding region of native PSEN2 not present in the PSEN2-encoding plasmids (forward primer=SEQ ID NO:88; probe=SEQ ID NO: 89; reverse primer=SEQ ID NO:90). The real-time PCR data was normalized to a house keeping gene whose expression was constant. Plasmids lacking either (a) the miRNA targeting sequences and siRNA sequences (e.g., hPSEN1v1.5, FIG. 15), or (b) the miRNA recognition sequences, siRNA sequences and any PSEN coding sequences (empty vector (“EV”), FIG. 15), were used as controls.

FIG. 15 shows the results of this experiment using plasmids that harbored the codon-optimized human PSEN1 coding sequence. Endogenous PSEN1 mRNA was detected and amplified with the forward primer SEQ ID NO:82; probe SEQ ID NO: 83; and reverse primer SEQ ID NO:84. Exogenous PSEN1 mRNA was detected and amplified with the forward primer SEQ ID NO:85; probe SEQ ID NO: 86; and reverse primer SEQ ID NO:87. In FIG. 15 the results have been normalized to the endogenous and exogenous levels detected when cells were transformed with a plasmid lacking any miRNA targeting sequences and siRNA sequences, but encoding a codon-optimized human PSEN1 (hPSEN1v1.5).

FIGS. 16A and 16B show the results of this experiment using plasmids that harbored the human PSEN2 coding sequence. Endogenous PSEN2 mRNA was detected and amplified with the forward primer SEQ ID NO:88; probe SEQ ID NO: 89; and reverse primer SEQ ID NO:90. Exogenous mRNA transcripts were detected and amplified with the forward primer SEQ ID NO:85; probe SEQ ID NO: 86; and reverse primer SEQ ID NO:87. In FIG. 16A the results have been normalized to the endogenous and exogenous levels detected

EXAMPLE 8
Sequences

SEQ ID NO: 1

GCAAAGGCTGTTGTCACTTGC

SEQ ID NO: 2

ATAGAAATACTGTTTCACAGAAAACAA

SEQ ID NO: 3

TCATCTTCTTCCTCATCTTGCTC

SEQ ID NO: 4

ACAAAGAGCATGATCACATGC

SEQ ID NO: 5

ATCATGATGGCAGCATTCAGAATTGAG

SEQ ID NO: 6

ACAATGACACTGATCATGATGGC

SEQ ID NO: 7

TAGTCATGACAACAATGACACTGATCA

SEQ ID NO: 8

TTTCTCTCCTGAGCTGTTTCAAC

SEQ ID NO: 9

CTTCTGCCATATTCACCAACCAC

SEQ ID NO: 10

GGAAAGTTCCTGGACAGCAGCTC

SEQ ID NO: 11

GGTTGTGTTCCAGTCTCCACTGGC

SEQ ID NO: 12

AAAGAAGAAACATCCATGGGATTCTAA

SEQ ID NO: 13

ATAGTCAAAGAAGAAACATCCAT

SEQ ID NO: 14

GTGTCCACATCTAACAAAGTCAAGATT

SEQ ID NO: 15

CAGTGAAATCGTCCTGTGACCACGCGT

SEQ ID NO: 16

TGTGACCACGCGTCAAGCTGCTGATGG

SEQ ID NO: 17

TTGACATCATTAGCTCACTGTATCCCC

SEQ ID NO: 18

TCCAAGTTGCTTAGAAAGCTTCTAC

SEQ ID NO: 19

ATATCTTACCAAGAATTTGAAAGGTAT

SEQ ID NO: 20

TTTCTTCATCAGTAAAATTCAGAGGGG

SEQ ID NO: 21

TTGGGAAAAGTCACTTTAGCTCTGTGG

SEQ ID NO: 22

CAGTGAATGGCGTGTAGATGAGCTGTC

SEQ ID NO: 23

TCAGTGAATGGCGTGTAGATGAGCTGT

SEQ ID NO: 24

CTCAGTGAATGGCGTGTAGATGAGCTG

SEQ ID NO: 25

TGGTCATAACCACGATGACGCTGATCA

SEQ ID NO: 26

AGCGGTACTTGTAGAGCACCACCAAGA

SEQ ID NO: 27

ACTGTCATAGGAGTCTTCTTCCATCTC

SEQ ID NO: 28

TATACAACTGCATCCAATGAAAATTCC

SEQ ID NO: 29

AAAACTATACAACTGCATCCAATGAAA

SEQ ID NO: 30

GTAAAACTATACAACTGCATCCAATGA

SEQ ID NO: 31

TTCTCAGTTCATCTGGATAAACCTGCT

SEQ ID NO: 32

TGATATTAGTATTAAGCCACTTCCGAG

SEQ ID NO: 33

GAATCCCATAGATACTTCTTCTTTAAATGTCCATACAAGAAGAAACATCCATGGGATTC

SEQ ID NO: 34

ACTTTTCATCAAATGCAGGTAAGGTTTACATTTTACAACTGCATCCAATGAAAATT

SEQ ID NO: 35

AGGCCTCTCTCTAGAATCCCATAGATACTTCTTCTTTAAATGTCCATACAAGAAGAAACATCCA

TGGGATTCGAATGGGGCTG

SEQ ID NO: 36

TGAGCTGTTGGATTAGTTTTCATCAAATGCAGGTAAGGTTTACATTTTACAACTGCATCCAATG

AAAATTTTCAGCTGCTTC

SEQ ID NO: 37

Example AAV-transgene containing AAV2 inverted terminal repeats, U6 promoter, 3 copies of hsa-pre-mir-124a-1-hPSEN1-1631-1652, CBA promoter, PSEN1 coding Sequence, rabbit polyadenylation Sequence, and AAV2 inverted terminal repeat.

SEQ ID NO: 38—Example AAV-transgene containing AAV2 inverted terminal repeats, U6 promoter, 3 copies of hsa-pre-mir-128a-hPSEN2-1766-1788, CBA promoter, PSEN2 coding SEQuence, rabbit polyadenylation SEQuence, and AAV2 inverted terminal repeat.

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTC

GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC

CTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACCAGGGTAATGAAGGTCGGGCAGGAA

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAA

TTAGAATTAATTTGAGTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATA

ATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTA

ACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCATGAGCT

GTTGGATTAGTTTTCATCAAATGCAGGTAAGGTTTACATTTTACAACTGCATCCAATGAAAATT

TTCAGCTGCTTCTGAGCTGTTGGATTACTTTTCATCAAATGCAGGTAAGGTTTACATTTTACAA

CTGCATCCAATGAAAATTTTCAGCTGCTTCTGAGCTGTTGGATTACTTTTCATCAAATGCAGGT

AAGGTTTACATTTTACAACTGCATCCAATGAAAATTTTCAGCTGCTTCTTTTTCGCGTCGACAT

TGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG

AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC

ATTGAGGTCAATAATGAGGTATGTTCCCATAGTAACGCCAATAGGGAGTTTCCATTGAGGTCAA

TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTA

CGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT

ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCA

CGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTT

TTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGG

GCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCG

AAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGG

GGCCACCATGCTCACATTCATGGCCTCTGACAGCGAGGAAGAAGTGTGTGATGAGCGGACGTCC

CTAATGTCGGCTGAGAGCCCCACGCCGCGCTCCTGCCAGGAGGGCAGGCAGGGCCCAGAGGATG

GAGAGAACACTGCCCAGTGGAGAAGCCAGGAGAACGAGGAGGACGGTGAGGAGGACCCTGACCG

CTATGTCTGTAGTGGGGTTCCCGGGCGGCCGCCAGGCCTGGAGGAAGAGCTGACCCTCAAATAC

GGAGCGAAGCACGTGATCATGCTGTTTGTGCCTGTCACTCTGTGCATGATCGTGGTGGTAGCCA

CCATCAAGTCTGTGCGCTTCTACACAGAGAAGAATGGACAGCTCATCTACACGCCATTCACTGA

GGACACACCCTCGGTGGGCCAGCGCCTCCTCAACTCCGTGCTGAACACCCTCATCATGATCAGC

GTCATCGTGGTTATGACCATCTTCTTGGTGGTGCTCTACAAGTACCGCTGCTACAAGTTCATCC

ATGGCTGGTTGATCATGTCTTCACTGATGCTGCTGTTCCTCTTCACCTATATCTACCTTGGGGA

AGTGCTCAAGACCTACAATGTGGCCATGGACTACCCCACCCTCTTGCTGACTGTCTGGAACTTC

GGGGCAGTGGGCATGGTGTGCATCCACTGGAAGGGCCCTCTGGTGCTGCAGCAGGCCTACCTCA

TCATGATCAGTGCGCTCATGGCCCTAGTGTTCATCAAGTACCTCCCAGAGTGGTCCGCGTGGGT

CATCCTGGGCGCCATCTCTGTGTATGATCTCGTGGCTGTGCTGTGTCCCAAAGGGCCTCTGAGA

ATGCTGGTAGAAACTGCCCAGGAGAGAAATGAGCCCATATTCCCTGCCCTGATATACTCATCTG

CCATGGTGTGGACGGTTGGCATGGCGAAGCTGGACCCCTCCTCTCAGGGTGCCCTCCAGCTCCC

CTACGACCCGGAGATGGAAGAAGACTCCTATGACAGTTTTGGGGAGCCTTCATACCCCGAAGTC

TTTGAGCCTCCCTTGACTGGCTACCCAGGGGAGGAGCTGGAGGAAGAGGAGGAAAGGGGCGTGA

AGCTTGGCCTCGGGGACTTCATCTTCTACAGTGTGCTGGTGGGCAAGGCGGCTGCCACGGGCAG

CGGGGACTGGAATACCACGCTGGCCTGCTTCGTGGCCATCCTCATTGGCTTGTGTCTGACCCTC

CTGCTGCTTGCTGTGTTCAAGAAGGCGCTGCCCGCCCTCCCCATCTCCATCACGTTCGGGCTCA

TCTTTTACTTCTCCACGGACAACCTGGTGCGGCCGTTCATGGACACCCTGGCCTCCCATCAGCT

CTACATCTGACATAGTCGACCCCTATCCATCACACTGGCGGCCGCTCGAGGACGGGGTGAACTA

CGCCTGAGGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAG

CATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGT

GTCTCTCACTCGGAAGCAATTCGTTGATCTGAATTTCGACCACCCATAATACCCATTACCCTGG

TAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCAC

TCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGC

TTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

-NM_000021.4 Homo sapiens presenilin 1 (PSEN1), coding sequence

SEQ ID NO: 39

ATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACC

TGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAG

CCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAG

CAAGATGAGGAAGAAGATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCT

TTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATAC

CCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGA

GCCCTGCACTCAATTCTGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCC

TGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATATCATCTCT

ATTGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCTATAACGTTGCT

GTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTC

ACTGGAAAGGTCCACTTCGACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCT

GGTGTTTATCAAGTACCTCCCTGAATGGACTGCGTGGCTCATCTTGGCTGTGATTTCAGTATAT

GATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGTATGCTGGTTGAAACAGCTCAGGAGA

GAAATGAAACGCTTTTTCCAGCTCTCATTTACTCCTCAACAATGGTGTGGTTGGTGAATATGGC

AGAAGGAGACCCGGAAGCTCAAAGGAGAGTATCCAAAAATTCCAAGTATAATGCAGAAAGCACA

GAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAG

CCCAGAGGGACAGTCATCTAGGGCCTCATCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGA

ACTTTCCAGCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGA

GATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACA

CAACCATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTGCCTTACATTATTACTCCTTGCCAT

TTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCC

ACAGATTATCTTGTAGAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCTAG

-NM_000447.3 Homo sapiens presenilin 2 (PSEN2), coding Sequence

SEQ ID NO: 40

ATGCTCACATTCATGGCCTCTGACAGCGAGGAAGAAGTGTGTGATGAGCGGACGTCCCTAATGT

CGGCTGAGAGCCCCACGCCGCGCTCCTGCCAGGAGGGCAGGCAGGGCCCAGAGGATGGAGAGAA

CACTGCCCAGTGGAGAAGCCAGGAGAACGAGGAGGACGGTGAGGAGGACCCTGACCGCTATGTC

TGTAGTGGGGTTCCCGGGCGGCCGCCAGGCCTGGAGGAAGAGCTGACCCTCAAATACGGAGCGA

AGCACGTGATCATGCTGTTTGTGCCTGTCACTCTGTGCATGATCGTGGTGGTAGCCACCATCAA

GTCTGTGCGCTTCTACACAGAGAAGAATGGACAGCTCATCTACACGCCATTCACTGAGGACACA

CCCTCGGTGGGCCAGCGCCTCCTCAACTCCGTGCTGAACACCCTCATCATGATCAGCGTCATCG

TGGTTATGACCATCTTCTTGGTGGTGCTCTACAAGTACCGCTGCTACAAGTTCATCCATGGCTG

GTTGATCATGTCTTCACTGATGCTGCTGTTCCTCTTCACCTATATCTACCTTGGGGAAGTGCTC

AAGACCTACAATGTGGCCATGGACTACCCCACCCTCTTGCTGACTGTCTGGAACTTCGGGGCAG

TGGGCATGGTGTGCATCCACTGGAAGGGCCCTCTGGTGCTGCAGCAGGCCTACCTCATCATGAT

CAGTGCGCTCATGGCCCTAGTGTTCATCAAGTACCTCCCAGAGTGGTCCGCGTGGGTCATCCTG

GGCGCCATCTCTGTGTATGATCTCGTGGCTGTGCTGTGTCCCAAAGGGCCTCTGAGAATGCTGG

TAGAAACTGCCCAGGAGAGAAATGAGCCCATATTCCCTGCCCTGATATACTCATCTGCCATGGT

GTGGACGGTTGGCATGGCGAAGCTGGACCCCTCCTCTCAGGGTGCCCTCCAGCTCCCCTACGAC

CCGGAGATGGAAGAAGACTCCTATGACAGTTTTGGGGAGCCTTCATACCCCGAAGTCTTTGAGC

CTCCCTTGACTGGCTACCCAGGGGAGGAGCTGGAGGAAGAGGAGGAAAGGGGCGTGAAGCTTGG

CCTCGGGGACTTCATCTTCTACAGTGTGCTGGTGGGCAAGGCGGCTGCCACGGGCAGCGGGGAC

TGGAATACCACGCTGGCCTGCTTCGTGGCCATCCTCATTGGCTTGTGTCTGACCCTCCTGCTGC

TTGCTGTGTTCAAGAAGGCGCTGCCCGCCCTCCCCATCTCCATCACGTTCGGGCTCATCTTTTA

CTTCTCCACGGACAACCTGGTGCGGCCGTTCATGGACACCCTGGCCTCCCATCAGCTCTACATC

TGA

>PSEN1 codon modified to avoid shRNAs

SEQ ID NO: 41

CCCAGATCTGCCACCATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGT

CTGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCA

CAACGACAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCC

CGGCAGGTGGTGGAACAGGACGAAGAGGAGGACGAAGAGCTGACATTGAAATATGGCGCCAAAC

ACGTCATAATGCTATTCGTGCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTC

AGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAG

ACTGTGGGCCAGAGAGCCCTGCATAGCATACTCAAGGCAGCTATTATGATTTCCGTGATCGTTG

TCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCT

TATTATATCATCTCTATTGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAA

ACCTATAACGTTGCTGTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTTGGTGTGGTGG

GAATGATTTCCATTCACTGGAAAGGTCCACTTCGACTCCAGCAGGCATATCTCATTATGATTAG

TGCCCTCATGGCCCTGGTGTTTATCAAGTACCTCCCTGAATGGACTGCGTGGCTCATCTTGGCT

GTGATTTCAGTATATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGAATGCTAGTCG

AGACGGCACAAGAAAGAAATGAAACGCTTTTTCCAGCTCTCATTTACTCCTCAACTATGGTATG

GCTAGTCAACATGGCAGAAGGAGACCCGGAAGCTCAAAGGAGAGTATCCAAAAATTCCAAGTAT

AATGCAGAAAGCACAGAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGCGGGTTCA

GTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCCTCATCGCTCTACACCTGAGTCACG

AGCAGCGGTGCAAGAGCTGTCTTCCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTA

AAACTTGGATTGGGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCGA

GTGGTGATTGGAATACTACGATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTGCCTTACATT

ATTACTCCTTGCCATTTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCACCTTTGGGCTT

GTTTTCTACTTTGCCACAGATTATCTTGTAGAGCCTTTTATGGACCAATTAGCATTCCATCAAT

TTTATATCTAGCATAGTCGACCCC

SEQ ID NO: 42

GAAATCACAGCCAAGATGAGC

SEQ ID NO: 43

ATGGAATGCTAATTGGTCCAT

SEQ ID NO: 44

GGAGCAAGATGAGGAAGAAGACGAATCTTCTTCCTCATCTTGCTCCTT

SEQ ID NO: 45

GCTCATCTTGGCTGTGATTTCCGAAGAAATCACAGCCAAGATGAGCTT

SEQ ID NO: 46

AAAGTTCCTGGACAGCAGCTCCGAAGAGCTGCTGTCCAGGAACTTTTT

SEQ ID NO: 47

GATGGAATGCTAATTGGTCCATCGAAATGGACCAATTAGCATTCCATT

>Human PSEN1 with all tolerant, non-preferred codons changed to

highly preferred synonymous codon. Changed codons in lower case:

SEQ ID NO: 48

ATGACAGAGTTACCTGCAcctTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACC

TGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAG

CctgGGCCACCCTGAGCCActgTCTAATGGAagaCCCCAGGGTAACTCCCGGCAGGTGGTGGAG

cagGATGAGGAAGAAGATGAGGAGCTGACActgAAATATGGCGCCAAGcacGTGATCATGCTCT

TTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATAC

CCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGA

GCCCTGCACTCAATTCTGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCC

TGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGctgATTATATCATCTct

gTTGctgCTGTTCTTTTTTTCATTCATTTACctgGGGGAAGTGTTTAAAACCTATAACGTTGCT

GTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTTggcGTGGTGGGAATGATTTCCATTC

ACTGGAAAggcCCActgagaCTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCT

GGTGTTTATCAAGTACCTCCCTGAATGGACTgccTGGCTCATCTTGGCTGTGATTTCAGTGTAT

GATTTAGTGGCTGTTctgTGTcctAAAGGTCCActgCGTATGCTGgtgGAAACAGCTCAGGAGA

GAAATGAAaccctgTTTCCAGCTCTCATTTACTCCTCAACAATGGTGTGGctgGTGAATATGGC

AGAAGGAGACcctGAAGCTCAAAGGAGAgtgTCCAAAAATTCCAAGTATAATGCAGAAAGCACA

GAAAGGGAGTCAcagGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAG

CCCAGAGGGACAGTcacctgGGGCCTcacCGCTCTACACCTGAGTCAagaGCTGCTGTCCAGGA

ActgTCCAGCAGTATCCTCGCTggcGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGA

GATTTCATTTTCTACAGTGTTCTGGTTggcAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACA

CAACCATAGCCTGTTTCGTAGCCatcTTAATTggcctgTGCCTTACActgctgCTCctgGCCAT

TTTCAAGAAAGCActgCCAGCTctgCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCC

ACAGATTATctggtgCAGCCTTTTATGGACcagctgGCATTCcaccagTTTTATATCtaaATGA

CAGAGTTACCTGCAcctTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACCTGAG

CAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAGCctg

GGCCACCCTGAGCCActgTCTAATGGAagaCCCCAGGGTAACTCCCGGCAGGTGGTGGAGcagG

ATGAGGAAGAAGATGAGGAGCTGACActgAAATATGGCGCCAAGcacGTGATCATGCTCTTTGT

CCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATACCCGG

AAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCC

TGCACTCAATTCTGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCCTGGT

GGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGctgATTATATCATCTctgTTG

ctgCTGTTCTTTTTTTCATTCATTTACctgGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGG

ACTACATTACTGTTGCACTCCTGATCTGGAATTTTggcGTGGTGGGAATGATTTCCATTCACTG

GAAAggcCCActgagaCTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTG

TTTATCAAGTACCTCCCTGAATGGACTgccTGGCTCATCTTGGCTGTGATTTCAGTGTATGATT

TAGTGGCTGTTctgTGTcctAAAGGTCCActgCGTATGCTGgtgGAAACAGCTCAGGAGAGAAA

TGAAaccctgTTTCCAGCTCTCATTTACTCCTCAACAATGGTGTGGctgGTGAATATGGCAGAA

GGAGACcctGAAGCTCAAAGGAGAgtgTCCAAAAATTCCAAGTATAATGCAGAAAGCACAGAAA

GGGAGTCAcagGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAGCCCA

GAGGGACAGTcacctgGGGCCTcacCGCTCTACACCTGAGTCAagaGCTGCTGTCCAGGAActg

TCCAGCAGTATCCTCGCTggcGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGAGATT

TCATTTTCTACAGTGTTCTGGTTggcAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACACAAC

CATAGCCTGTTTCGTAGCCatcTTAATTggcctgTGCCTTACActgctgCTCctgGCCATTTTC

AAGAAAGCActgCCAGCTctgCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAG

ATTATctggtgCAGCCTTTTATGGACcagctgGCATTCcaccagTTTTATATCtaa

Example AAV-transgene containing AAV2 inverted terminal repeats,

U6 promoter, 1 copy of Anti-hPSENl-401-421 (underlined), H1

promoter, 1 copy of Anti-hPSEN1-953-973 (underlined), GAG

promoter, codon modified PSEN1 coding

SEQuence, rabbit polyadenylation

SEQuence, and AAV2 inverted terminal repeat.

SEQ ID NO: 49

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTC

GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC

CTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACCAGGGTAATGGGGATCCTCTAGAAC

TATGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC

GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACA

AAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAA

ATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGT

GGAAAGGACGAAACACCGGAGCAAGATGAGGAAGAAGACGAATCTTCTTCCTCATCTTGCTCCT

TTTTTCTAGAGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGG

CGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGC

CCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGA

TTTGGGAATCTTATAAGTTCTGTATGAGACCACGCTCATCTTGGCTGTGATTTCCGAAGAAATC

ACAGCCAAGATGAGCTTTTTTCTAGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAAT

TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC

CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAG

TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTT

GGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG

CCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG

TATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTC

CCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGG

GCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA

GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAG

GCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGC

CTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGT

TACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTA

ATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTG

TGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCT

CCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGT

GCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAA

GGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGC

AACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCG

TACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGC

GGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCG

GCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCA

GGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCT

AGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTG

CGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTG

CCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCC

TCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTG

TGCTGTCTCATCATTTTGGCAAAGAATTCACGCCCCAGATCTGCCACCATGACAGAGTTACCTG

CACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACCTGAGCAATACTGTACG

TAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAGCCTTGGCCACCCTGAG

CCATTATCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAACAGGACGAAGAGGAGG

ATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCTTTGTCCCTGTGACTCT

CTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATACCCGGAAGGATGGGCAG

CTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTC

TGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAA

ATACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATATCATCTCTATTGTTGCTGTTCTTT

TTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGGACTACATTACTG

TTGCACTCCTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACT

TCGACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCAAGTAC

CTCCCTGAATGGACTGCGTGGCTGATTCTAGCCGTAATCTCAGTATATGATTTAGTGGCTGTTT

TGTGTCCGAAAGGTCCACTTCGTATGCTGGTTGAAACAGCTCAGGAGAGAAATGAAACGCTTTT

TCCAGCTCTCATTTACTCCTCAACAATGGTGTGGTTGGTGAATATGGCAGAAGGAGACCCGGAA

GCTCAAAGGAGAGTATCCAAAAATTCCAAGTATAATGCAGAAAGCACAGAAAGGGAGTCACAAG

ACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCA

TCTAGGGCCTCATCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCAGTATC

CTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGAGATTTCATTTTCTACA

GTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACACAACCATAGCCTGTTT

CGTAGCCATATTAATTGGTTTGTGCCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTG

CCAGCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAGATTATCTTGTAC

AGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCTAGCATAGTCGACCCCTATCCAT

CACACTGGCGGCCGCTCGAGGACGGGGTGAACTACGCCTGAGGATCCGATCTTTTTCCCTCTGC

CAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTT

ATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGCAATTCGTTGATCT

GAATTTCGACCACCCATAATACCCATTACCCTGGTAGATAAGTAGCATGGCGGGTTAATCATTA

ACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGA

GGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA

GCGCGCAG

>CAG promoter

SEQ ID NO: 50

GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA

TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC

CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGAC

GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG

ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCG

AGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCCCCCCCCA

GGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAAAGGTGCGGCGGCAGCCAATC

AGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA

GCGAAGCGCGCGGCGGGCGGGAGTCGTTGCGCGCTGCCTTCCCCCCGTGCCCCGCTCCGCCGCC

GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACG

GCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCT

GCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCG

TGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCT

GCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGG

TGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGG

GGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAG

TTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGT

GCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAG

GGCTCGGGGGAAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGC

AGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTG

CGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGG

CGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTC

CCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGG

GGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTT

CTTTTTCCTACAG

>CBA promoter.

SEQ ID NO: 51

TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTT

GTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCG

CCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCA

ATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAA

AAAGCGAAGCGCGCGGCGGGCG

>UBC promoter

SEQ ID NO: 52

GGTGCAGCGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGC

GCTGCCACGTCAGACGAAGGGCGCAGGAGCGTTCCTGATCCTTCCGCCCGGACGCTCAGGACAG

CGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACG

GGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAG

TCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGG

ACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGT

GGATCGCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCG

CCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCG

AGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCACAAAATGGCGGCTGTTCCCGAGTCTT

GAATGGAAGACGCTTGTAAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGC

GGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGG

CTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGGTTTGTCG

TCTGGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGC

GCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGC

CGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCT

CCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGG

TCGGTTTTATGTACCTATCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGT

TGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATG

TAATTTTGAGTGTTAGACTAGTAAA

>PGK promoter

SEQ ID NO: 53

TTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGC

TGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCG

CCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAG

TTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTA

GTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCA

ATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGG

CTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCA

CGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCT

>Ef1α promoter

SEQ ID NO: 54

GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGG

GGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTG

TACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGA

ACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGG

GCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCA

GTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCT

TAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGC

GAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTT

TTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTG

CACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACAT

GTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGG

CCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTG

GCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCT

CAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGC

CTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTC

GATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGG

AGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTC

CTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAA

AGTTTTTTTCTTCCATTTCAGGTGTCGTGA

>CMV promoter

SEQ ID NO: 55

GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCC

ATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC

CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATT

GACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT

GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC

ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG

TGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG

TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA

TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATA

TAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGAC

TCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTT

>NSE promoter

SEQ ID NO: 56

AGCTCTGAGCTCCTCCTCTGCTCGCCCAATCCTTCCAACCCCCTATGGTGGTATGGCTGACACA

GAAAATGTCTGCTCCTGTATGGGACATTTGCCCCTCTTCTCCAAATATAAGACAGGATGAGGCC

TAGCTTTTGCTGCTCCAAAGTTTTAAAAGAACACATTGCACGGCATTTAGGGACTCTAAAGGGT

GGAGGAGGAATGAGGGAATTGCATCATGCCAAGGCTGGTCCTCATCCATCACTGCTTCCAGGGC

CCAGAGTGGCTTCCAGGAAGTATTCTTACAAAGGAAGCCCGATCTGTAGCTAACACTCAGAGCC

CATTTTCCTGCGTTAACCCCTCCCGACCTCATATACAGGAGTAACATGATCAGTGACCTGGGGG

AGCTGGCCAAACTGCGGGACCTGCCCAAGCTGAGGGCCTTGGTGCTGCTGGACAACCCCTGTGC

CGATGAGACTGACTACCGCCAGGAGGCCCTGGTGCAGATGGCACACCTAGAGCGCCTAGACAAA

GAGTACTATGAGGACGAGGACCGGGCAGAAGCTGAGGAGATCCGACAGAGGCTGAAGGAGGAAC

AGGAGCAAGAACTCGACCCGGACCAAGACATGGAACCGTACCTCCCGCCAACTTAGTGGCTCCT

CTAGCCTGCAGGGACAGTAAAGGTGATGGCAGGAAGGCAGCCCCCGGAGGTCAAAGGCTGGGCA

CGCGGGAGGAGAGGCCAGAGTCAGAGGCTGCGGGTATCTCAGATATGAAGGAAAGATGAGAGAG

GCTCAGGAAGAGGTAAGAAAAGACACAAGAGACCAGAGAAGGGAGAAGAATTAGAGAGGGAGGC

AGAGGACCGCTGTCTCTACAGACATAGCTGGTAGAGACTGGGAGGAAGGGATGAACCCTGAGCG

CATGAAGGGAAGGAGGTGGCTGGTGGTATATGGAGGATGTAGCTGGGCCAGGGAAAAGATCCTG

GAGTAAAAATCTGAAGCTAAAAATAACAGGACACGGGGTGGAGAGGCGAAAGGAGGGCAGAGTG

AGGCAGAGAGACTGAGAGGCCTGGGGATGTGGGCATTCCGGTAGGGCACACAGTTCACTTGTCT

TCTCTTTTTCCAGGAGGCCAAAGATGCTGACGTCAAGAACTCATAATACCCCAGTGGGGACCAC

CGCATTCATAGCCCTGTTACAAGAAGTGGGAGATGTTCCTTTTTGTCCCAGACTGGAAATCCGT

TACATCCCGAGGCTCAGGTTCTGTGGTGGTCATCTCTGTGTGGCTTGTTCTGTGGGCCTACCTA

AAGTCCTAAGCACAGCTCTCAAGCAGATCCGAGGCGACTAAGATGCTAGTAGGGGTTGTCTGGA

GAGAAGAGCCGAGGAGGTGGGCTGTGATGGATCAGTTCAGCTTTCAAATAAAAAGGCGTTTTTA

TATTCTGTGTCGAGTTCGTGAACCCCTGTGGTGGGCTTCTCCATCTGTCTGGGTTAGTACCTGC

CACTATACTGGAATAAGGGGACGCCTGCTTCCCTCGAGTTGGCTGGACAAGGTTATGAGCATCC

GTGTACTTATGGGGTTGCCAGCTTGGTCCTGGATCGCCCGGGCCCTTCCCCCACCCGTTCGGTT

CCCCACCACCACCCGCGCTCGTACGTGCGTCTCCGCCTGCAGCTCTTGACTCATCGGGGCCCCC

GGGTCACATGCGCTCGCTCGGCTCTATAGGCGCCGCCCCCTGCCCACCCCCCGCCCGCGCTGGG

AGCCGCAGCCGCCGCCACTCCTGCTCTCTCTGCGCCG

>MeCP2 promoter

SEQ ID NO: 57

TGCCCATTATAAACGTCTGCAAAGACCAAGGTTTGATATGTTGATTTTACTGTCAGCCTTAAGA

GTGCGAGATCTGCTAATTTAGTGTAATAATACAATGAGTAGACCCTTTAAAACAAGTCCCTTGG

CTTGGAACAACGCCAGGCTCCTCAACAGGCAACTTTGCTAGTTCTACAGAAAATGATAATAAAG

AAATGCTGGTGAAGTCAAATGCTTATCACAATGGTGAACTACTCAGCAGGGAGGCTCTAATAGG

CGCCAAGAGCCTAGACTTCCTTAAGCGCCAGAGTCCACAAGGGCCCAGTTAATCCTCAACATTC

AAATGCTGCCCACAAAACCAGCCCCTCTGTGCCCTAGCCGCCTCTTTTTTCCAAGTGACAGTAG

AACTCCACCAATCCGCAGCTGAATGGGGTCCGCCTCTTTTCCCTGCCTAAACAGACAGGAACTC

CTGCCAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGG

TAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTC

GGGAGGGCGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTTGCGCGCGCGCTCCCTCC

TCTCGGAGAGAGGGCTGTGGTAAAACCCGTCCGGAAAATGGCCGCCGCTGCCGCCACCGCCGCC

GCCGCCGCCGCGCCGAGCGGAGGAGGAGG

>GFAP promoter

SEQ ID NO: 58

GGCAACATGGCAAGACCCTATCTCTACAAAAAAAGTTAAAAAATCAGCCACGTGTGGTGACACA

CACCTGTAGTCCCAGCTATTCAGGAGGCTGAGGTGAGGGGATCACTTAAGGCTGGGAGGTTGAG

GCTGCAGTGAGTCGTGGTTGCGCCACTGCACTCCAGCCTGGGCAACAGTGAGACCCTGTCTCAA

AAGACAAAAAAAAAAAAAAAAAAAAAAAGAACATATCCTGGTGTGGAGTAGGGGACGCTGCTCT

GACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGGAGGCAGACAGCCAGGCCTTG

TCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGGGCCTCCTCTTCATGCCCAGTG

AATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACAGTGCCTGCTTCCCGCCGCACC

CCAGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCC

TGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGC

GCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGC

CCAGGCATGGACAGTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGG

ACACAAATGGGTGAGGGGACTGGGCAGGGTTCTGACCCTGTGGGACCAGAGTGGAGGGCGTAGA

TGGACCTGAAGTCTCCAGGGACAACAGGGCCCAGGTCTCAGGCTCCTAGTTGGGCCCAGTGGCT

CCAGCGTTTCCAAACCCATCCATCCCCAGAGGTTCTTCCCATCTCTCCAGGCTGATGTGTGGGA

ACTCGAGGAAATAAATCTCCAGTGGGAGACGGAGGGGTGGCCAGGGAAACGGGGCGCTGCAGGA

ATAAAGACGAGCCAGCACAGCCAGCTCATGTGTAACGGCTTTGTGGAGCTGTCAAGGCCTGGTC

TCTGGGAGAGAGGCACAGGGAGGCCAGACAAGGAAGGGGTGACCTGGAGGGACAGATCCAGGGG

CTAAAGTCCTGATAAGGCAAGAGAGTGCCGGCCCCCTCTTGCCCTATCAGGACCTCCACTGCCA

CATAGAGGCCATGATTGACCCTTAGACAAAGGGCTGGTGTCCAATCCCAGCCCCCAGCCCCAGA

ACTCCAGGGAATGAATGGGCAGAGAGCAGGAATGTGGGACATCTGTGTTCAAGGGAAGGACTCC

AGGAGTCTGCTGGGAATGAGGCCTAGTAGGAAATGAGGTGGCCCTTGAGGGTACAGAACAGGTT

CATTCTTCGCCAAATTCCCAGCACCTTGCAGGCACTTACAGCTGAGTGAGATAATGCCTGGGTT

ATGAAATCAAAAAGTTGGAAAGCAGGTCAGAGGTCATCTGGTACAGCCCTTCCTTCCCTTTTTT

TTTTTTTTTTTTGTGAGACAAGGTCTCTCTCTGTTGCCCAGGCTGGAGTGGCGCAAACACAGCT

CACTGCAGCCTCAACCTACTGGGCTCAAGCAATCCTCCAGCCTCAGCCTCCCAAAGTGCTGGGA

TTACAAGCATGAGCCACCCCACTCAGCCCTTTCCTTCCTTTTTAATTGATGCATAATAATTGTA

AGTATTCATCATGGTCCAACCAACCCTTTCTTGACCCACCTTCCTAGAGAGAGGGTCCTCTTGC

TTCAGCGGTCAGGGCCCCAGACCCATGGTCTGGCTCCAGGTACCACCTGCCTCATGCAGGAGTT

GGCGTGCCCAGGAAGCTCTGCCTCTGGGCACAGTGACCTCAGTGGGGTGAGGGGAGCTCTCCCC

ATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACC

CGGGCATCGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCA

GAGCAGG

>GUSB promoter

SEQ ID NO: 59

GAATTCCTGCTGGGAAAAGCAAGTGGAGGTGCTCCTTGAAGAAACAGGGGGATCCCACCGATCT

CAGGGGTTCTGTTCTGGCCTGCGGCCCTGGATCGTCCAGCCTGGGTCGGGGTGGGGAGCAGACC

TCGCCCTTATCGGCTGGGGCTGAGGGTGAGGGTCCCGTTTCCCCAAAGGCCTAGCCTGGGGTTC

CAGCCACGAAGCCCTACCGGGAGCGCCCGGCCCCGCCCCTCCAGGCCTGGCACTCGTCCTCAAC

CAAGATGGCGCGGATGGCTTCAGGCGCATCACGACACCGGCGCGTCACGCGACCCGCCCTACGG

GCACCTCCCGCGCTTTTCTTAGCGCCGCAGACGGTGGCCGAGCGGGGGACCGGGAAGC

>RSV promoter

SEQ ID NO: 60

AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCC

TTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCT

TATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAG

AGATATTGTATTTAAGTGCCTAGCTCGATACATAAAC

SV40promoter

SEQ ID NO: 61

GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTC

AGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTC

AATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTT

CCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTC

GGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGAT

CGATCAAGAGACAGGATGAGGATCGTTTCGC

NPY promoter

SEQ ID NO: 62

TTTTGGCCAGGGGATGTGGCTTGGACTGGAGAGAAAGGAGATAAGGATGTAAACACATGTAGGG

CATATCACCCCCTATTTTTTATTCTCTGAATCCTTAACCCTCAGAATAAGTTCTTATTCTTGAG

AATCAATGACATTATCTTAAGCTAAATTAATCAAGCCTCCACAGTGTTCTTCTCTCAATAGTGG

TGTGGGCCTTCCTAGAAGTAATTTTTCCCAAATTCAGTGATACATTTTAAGTTCAGATTTTAAT

TGATATGAATCTGTGATACACTCTAAAATAAGATTATTTTATTGAAAAGTGGACTGTAACTTTC

CCTTTATCTAGGAAGAGCTCTAAGTTAGAAGATGTTTTGCACTTTTACCGAAGGCTGTGTCTTG

TAAGCACCCCCGAGCAACTCTGAGAGCCTTGATTTTTGTGTCCTCAGCATATGTTTGTGTAATA

CAGAAAGAGAAGCAGTTGCCAAGTGAAAGGGATGTTGGTCTCCAAAATTATAGTTTGATCCCAC

AAACACACAAACACATACATGCAAAGGATTGTTTGCTTCACGGTTTTTGATATTTAATTCAATG

CTGTTGGAACAGCACAAAAACTAAGTGTCAGTTTAACAGAATCACTTGTCCTTTTAGCATTAAA

ATAACATGGAACTTAATGCTTTAATTTCCCAACATGCCTTTTTATTTAGAAAGATTCAGACTTT

TATTTCATTTAGAAATAAAATGCCATTTTATTTAGAAAGATACAGGAGCATTCATTCACGGAAC

TTTCAGATCTCAGTCCACTGCATAAAATCTTGATCCTGTAATAATAGTTTCTGTATCTTGCATA

TTCATTCAACAGGTTTAACGCGATGAGCAAATTAATGTTCATCGTTTTTAACATGTTTCGTCTT

AATCAGAACCCACATTCTCAACGTTAATTGAACGTAGATAGGACTATACAAGGGTTAGTAAATA

AGACAGAAACTGTTGCTCATTTAACCACCGTCACTTTGGA

SST promoter

SEQ ID NO: 63

ACACTAAAATGTTAGAGTATGATGACAGATGGAGTTGTCTGGGTACATTTGTGTGCATTTAAGG

GTGATAGTGTATTTGCTCTTTAAGAGCTGAGTGTTTGAGCCTCTGTTTGTGTGTAATTGAGTGT

GCATGTGTGGGAGTGAAATTGTGGAATGTGTATGCTCATAGCACTGAGTGAAAATAAAAGATTG

TATAAATCGTGGGGCATGTGGAATTGTGTGTGCCTGTGCGTGTGCAGTATTTTTTTTTTTTTAA

GTAAGCCACTTTAGATCTTGTCACCTCCCCTGTCTTCTGTGATTGATTTTGCGAGGCTAATGGT

GCGTAAAAGGGCTGGTGAGATCTGGGGGCGCCTCCTAGCCTGACGTCAGAGAGAGAGTTTAAAA

CAGAGGGAGACGGTTGAGAGCACACAAGCCGCTTTAGGAGCGAGGTTCGGAGCCATCGCTGCTG

CCTGCTGATCCGCGCCTAGAGTTTGACCAGCC

Synapsin promoter 1

SEQ ID NO: 64

ACACCACCCAAGTGTCCACCTCCGCTTGTCTGATGCTGTCTATGACGCCCCCGCTCTCTGCCTA

GCTGAGCCTGTGTGGATGTGGGAGACTAATCTCCCCGCGGGCACTGCGTGTGACCTCACCCCCC

TCTGTGAGGGGGTTATTTCTCTACTTTCGTGTCTCTGAGTGTGCTTCCAGTGCCCCCCTCCCCC

CAAAAAATGCCTTCTGAGTTGAATATCAACACTACAAACCGAGTATCTGCAGAGGGCCCTGCGT

ATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGAC

CCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGG

GGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCC

TTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTC

GCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAG

CCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGC

CGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAG

Synapsin promoter 2

SEQ ID NO: 65

ACACTACAAACCGAGTATCTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGG

ATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCC

CCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGC

GTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCAC

CGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCG

GCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGAT

AGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCG

GTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAG

>β-globin promoter

SEQ ID NO: 66

GCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATTAATTTTAACACCAAT

TCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAGAGACAGTGTTCTCTGCACA

GATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCA

CAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGG

TAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAG

GTAGGATCAGTTGCTCCTCACATTTGCTTCTGACATAGTTGTGTTG

(consensus Kozak sequence)

SEQ ID NO: 67

GCCGCCRCCAUGG

(pAT049)

SEQ ID NO: 68

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGAATTATGTTTCGAATTTGACTTTAGAGGTT

TACATTTCTAGTCAAAGAAGAAACATCCATTCAGCTGCTCCTTTTTTCCGGGACGCGTCAA

TTGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA

GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT

GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCC

AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA

GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGC

CCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA

CGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCC

CCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGG

GGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA

GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGC

GAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTC

GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAG

ACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAAC

GCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAA

AATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCT

CTTTCTTTGAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATA

ACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATA

TAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCAT

TCTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTT

TGCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTG

TGTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGT

CTCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATG

TTCCAGATTACGCTACAGAATTACCTGCCCCCTTGAGCTACTTCCAGAATGCACAGATGAG

CGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAACAGAGAACGGCAGGAA

CACAACGACAGGCGGAGCCTGGGCCACCCTGAGCCCCTGTCTAATGGAAGACCCCAGGGTA

ACAGCAGACAGGTGGTGGAACAAGATGAGGAAGAGGACGAGGAGCTGACCCTGAAGTACGG

CGCCAAGCACGTGATCATGCTCTTCGTGCCCGTGACTCTCTGCATGGTGGTGGTGGTGGCT

ACAATCAAGAGCGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCA

CAGAAGACACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATCCTGAATGCCGCCATCAT

GATCAGCGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTAT

AAGGTCATCCATGCCTGGCTGATCATATCATCTCTGTTGCTGCTGTTCTTTTTTAGCTTCA

TTTACCTGGGCGAAGTGTTTAAAACCTATAACGTTGCCGTGGACTACATTACTGTTGCCCT

CCTGATCTGGAACTTCGGCGTGGTGGGCATGATTTCCATTCACTGGAAAGGCCCCCTGAGA

CTGCAGCAGGCATACCTCATTATGATCTCCGCCCTCATGGCCCTGGTGTTCATCAAGTACC

TGCCCGAGTGGACTGCTTGGCTCATCTTGGCTGTGATCTCCGTGTATGATTTAGTGGCTGT

TCTGTGTCCTAAAGGTCCACTGCGTATGCTGGTGGAAACAGCTCAGGAAAGAAATGAAACA

CTGTTTCCTGCTCTGATTTACTCCTCAACAATGGTGTGGCTCGTGAATATGGCCGAAGGAG

ACCCTGAAGCCCAACGGAGAGTGTCCAAAAACTCCAAGTATAACGCCGAGAGCACAGAAAG

GGAGAGCCAGGATACAGTTGCCGAGAATGACGATGGCGGCTTCAGTGAGGAATGGGAAGCC

CAGAGGGACAGCCACCTGGGGCCTCACAGAAGCACCCCTGAGTCTAGAGCCGCTGTCCAGG

AACTGTCCAGCTCCATCCTGGCCGGCGAAGACCCCGAAGAAAGGGGAGTAAAACTTGGACT

GGGAGATTTCATCTTCTACAGTGTTCTCGTTGGCAAAGCCAGCGCAACAGCTAGCGGAGAC

TGGAACACAACAATAGCCTGTTTCGTAGCCATCTTAATTGGCCTGTGCCTTACACTTCTGC

TCCTGGCCATCTTCAAGAAGGCCCTGCCAGCCCTGCCTATCAGCATCACCTTCGGGCTTGT

TTTCTACTTTGCCACCGATTATCTGGTGCAGCCCTTCATGGACCAGCTGGCCTTCCACCAG

TTTTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACC

CCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCT

AATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGG

AGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCT

ATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTG

GGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACC

AGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGG

TCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACA

GGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCG

TCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATG

TTCCAAATTCGAAATTTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCC

CCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCC

CATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGGTGA

ATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAATAGGCAGAAATCGAGCCTCAA

AGAGCTTACAGTCTGGTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAG

GAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAG

GGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC

TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT

GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGT

ATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTAC

GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTA

CACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT

CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT

TTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGC

CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT

GTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATT

TTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT

TTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGC

CGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGT

CTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA

GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTT

ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAAT

GTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA

GACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAG

ACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCG

CTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGC

CGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC

GGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG

TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGG

CGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC

ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACC

AAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGA

TGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCG

AGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCA

TGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCG

CTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATC

GCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAG

ACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG

GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG

GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA

TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT

GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAA

AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT

CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT

TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTG

CCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

CAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC

GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG

TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA

CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT

ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG

GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC

GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC

TTTTGCTGGCCTTTTGCTCACATGT

(pAT050)

SEQ ID NO: 69

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGAGGTAGAAAGTAATAATTTCTTGGGTAGTTTGGAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGATAGCACATCTTTGAAAGTCATAAGAGGTT

TACATTTCTTGACTTTGTTAGATGTGGACTTCAGCTGCTTCTTTTTTCCGGGACGCGTCAA

TTGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA

GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT

GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCC

AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA

GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGC

CCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA

CGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCC

CCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGG

GGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA

GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGC

GAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTC

GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAG

ACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAAC

GCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAA

AATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCT

CTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATA

ACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATA

TAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCAT

TCTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTT

TGCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTG

TGTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGT

CTCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATG

TTCCAGATTACGCTACAGAATTACCTGCCCCCTTGAGCTACTTCCAGAATGCACAGATGAG

CGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAACAGAGAACGGCAGGAA

CACAACGACAGGCGGAGCCTGGGCCACCCTGAGCCCCTGTCTAATGGAAGACCCCAGGGTA

ACAGCAGACAGGTGGTGGAACAAGATGAGGAAGAGGACGAGGAGCTGACCCTGAAGTACGG

CGCCAAGCACGTGATCATGCTCTTCGTGCCCGTGACTCTCTGCATGGTGGTGGTGGTGGCT

ACAATCAAGAGCGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCA

CAGAAGACACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATCCTGAATGCCGCCATCAT

GATCAGCGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTAT

AAGGTCATCCATGCCTGGCTGATCATATCATCTCTGTTGCTGCTGTTCTTTTTTAGCTTCA

TTTACCTGGGCGAAGTGTTTAAAACCTATAACGTTGCCGTGGACTACATTACTGTTGCCCT

CCTGATCTGGAACTTCGGCGTGGTGGGCATGATTTCCATTCACTGGAAAGGCCCCCTGAGA

CTGCAGCAGGCATACCTCATTATGATCTCCGCCCTCATGGCCCTGGTGTTCATCAAGTACC

TGCCCGAGTGGACTGCTTGGCTCATCTTGGCTGTGATCTCCGTGTATGATTTAGTGGCTGT

TCTGTGTCCTAAAGGTCCACTGCGTATGCTGGTGGAAACAGCTCAGGAAAGAAATGAAACA

CTGTTTCCTGCTCTGATTTACTCCTCAACAATGGTGTGGCTCGTGAATATGGCCGAAGGAG

ACCCTGAAGCCCAACGGAGAGTGTCCAAAAACTCCAAGTATAACGCCGAGAGCACAGAAAG

GGAGAGCCAGGATACAGTTGCCGAGAATGACGATGGCGGCTTCAGTGAGGAATGGGAAGCC

CAGAGGGACAGCCACCTGGGGCCTCACAGAAGCACCCCTGAGTCTAGAGCCGCTGTCCAGG

AACTGTCCAGCTCCATCCTGGCCGGCGAAGACCCCGAAGAAAGGGGAGTAAAACTTGGACT

GGGAGATTTCATCTTCTACAGTGTTCTCGTTGGCAAAGCCAGCGCAACAGCTAGCGGAGAC

TGGAACACAACAATAGCCTGTTTCGTAGCCATCTTAATTGGCCTGTGCCTTACACTTCTGC

TCCTGGCCATCTTCAAGAAGGCCCTGCCAGCCCTGCCTATCAGCATCACCTTCGGGCTTGT

TTTCTACTTTGCCACCGATTATCTGGTGCAGCCCTTCATGGACCAGCTGGCCTTCCACCAG

TTTTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACC

CCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCT

AATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGG

AGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCT

ATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTG

GGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACC

AGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGG

TCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACA

GGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCG

TCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATG

TTCCAAATTCGAAATTTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCC

CCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCC

CATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGGTGA

ATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCTCAA

AGAGCTTACAGTCTGGTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAG

GAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAG

GGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC

TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT

GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGT

ATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTAC

GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTA

CACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT

CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT

TTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGC

CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT

GTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATT

TTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT

TTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGC

CGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGT

CTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA

GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTT

ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAAT

GTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA

GACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAG

ACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCG

CTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGC

CGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC

GGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG

TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGG

CGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC

ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACC

AAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGA

TGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCG

AGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCA

TGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCG

CTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATC

GCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAG

ACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG

GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG

GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA

TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT

GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAA

AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT

CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT

TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTG

CCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

CAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC

GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG

TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA

CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT

ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG

GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC

GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC

TTTTGCTGGCCTTTTGCTCACATGT

(pAT051)

SEQ ID NO: 70

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGAGACTAGAAAAGCCTTTTGACTACGAGGTT

TACATTTCGTTCAAAATCGTTTTCTATAGTTCAGCTGCTTCTTTTTTCCGGGACGCGTCAA

TTGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA

GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT

GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCC

AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA

GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGC

CCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA

CGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCC

CCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGG

GGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA

GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGC

GAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTC

GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAG

ACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAAC

GCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAA

AATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCT

CTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATA

ACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATA

TAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCAT

TCTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTT

TGCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTG

TGTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGT

CTCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATG

TTCCAGATTACGCTACAGAATTACCTGCCCCCTTGAGCTACTTCCAGAATGCACAGATGAG

CGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAACAGAGAACGGCAGGAA

CACAACGACAGGCGGAGCCTGGGCCACCCTGAGCCCCTGTCTAATGGAAGACCCCAGGGTA

ACAGCAGACAGGTGGTGGAACAAGATGAGGAAGAGGACGAGGAGCTGACCCTGAAGTACGG

CGCCAAGCACGTGATCATGCTCTTCGTGCCCGTGACTCTCTGCATGGTGGTGGTGGTGGCT

ACAATCAAGAGCGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCA

CAGAAGACACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATCCTGAATGCCGCCATCAT

GATCAGCGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTAT

AAGGTCATCCATGCCTGGCTGATCATATCATCTCTGTTGCTGCTGTTCTTTTTTAGCTTCA

TTTACCTGGGCGAAGTGTTTAAAACCTATAACGTTGCCGTGGACTACATTACTGTTGCCCT

CCTGATCTGGAACTTCGGCGTGGTGGGCATGATTTCCATTCACTGGAAAGGCCCCCTGAGA

CTGCAGCAGGCATACCTCATTATGATCTCCGCCCTCATGGCCCTGGTGTTCATCAAGTACC

TGCCCGAGTGGACTGCTTGGCTCATCTTGGCTGTGATCTCCGTGTATGATTTAGTGGCTGT

TCTGTGTCCTAAAGGTCCACTGCGTATGCTGGTGGAAACAGCTCAGGAAAGAAATGAAACA

CTGTTTCCTGCTCTGATTTACTCCTCAACAATGGTGTGGCTCGTGAATATGGCCGAAGGAG

ACCCTGAAGCCCAACGGAGAGTGTCCAAAAACTCCAAGTATAACGCCGAGAGCACAGAAAG

GGAGAGCCAGGATACAGTTGCCGAGAATGACGATGGCGGCTTCAGTGAGGAATGGGAAGCC

CAGAGGGACAGCCACCTGGGGCCTCACAGAAGCACCCCTGAGTCTAGAGCCGCTGTCCAGG

AACTGTCCAGCTCCATCCTGGCCGGCGAAGACCCCGAAGAAAGGGGAGTAAAACTTGGACT

GGGAGATTTCATCTTCTACAGTGTTCTCGTTGGCAAAGCCAGCGCAACAGCTAGCGGAGAC

TGGAACACAACAATAGCCTGTTTCGTAGCCATCTTAATTGGCCTGTGCCTTACACTTCTGC

TCCTGGCCATCTTCAAGAAGGCCCTGCCAGCCCTGCCTATCAGCATCACCTTCGGGCTTGT

TTTCTACTTTGCCACCGATTATCTGGTGCAGCCCTTCATGGACCAGCTGGCCTTCCACCAG

TTTTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACC

CCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCT

AATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGG

AGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCT

ATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTG

GGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACC

AGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGG

TCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACA

GGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCG

TCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATG

TTCCAAATTCGAAATTTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCC

CCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCC

CATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGGTGA

ATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAATAGGCAGAAATCGAGCCTCAA

AGAGCTTACAGTCTGGTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAG

GAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAG

GGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC

TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT

GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGT

ATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTAC

GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTA

CACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT

CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT

TTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGC

CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT

GTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATT

TTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT

TTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGC

CGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGT

CTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA

GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTT

ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAAT

GTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA

GACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAG

ACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCG

CTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGC

CGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC

GGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG

TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGG

CGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC

ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACC

AAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGA

TGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCG

AGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCA

TGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCG

CTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATC

GCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAG

ACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG

GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG

GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA

TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT

GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAA

AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT

CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT

TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTG

CCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

CAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC

GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG

TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA

CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT

ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG

GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC

GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC

TTTTGCTGGCCTTTTGCTCACATGT

(pAT052)

SEQ ID NO: 71

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGATTCTGGTCACTACACGATTCCTCGAGGTT

TACATTTCGAAATCGTCCTGTGACCACGCTTCAGCTGCTTCTTTTTTCCGGGACGCGTCAA

TTGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA

GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT

GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCC

AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA

GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGC

CCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA

CGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCC

CCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGG

GGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA

GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGC

GAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTC

GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAG

ACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAAC

GCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAA

AATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCT

CTTTCTTTGAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATA

ACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATA

TAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCAT

TCTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTT

TGCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTG

TGTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGT

CTCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATG

TTCCAGATTACGCTACAGAATTACCTGCCCCCTTGAGCTACTTCCAGAATGCACAGATGAG

CGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAACAGAGAACGGCAGGAA

CACAACGACAGGCGGAGCCTGGGCCACCCTGAGCCCCTGTCTAATGGAAGACCCCAGGGTA

ACAGCAGACAGGTGGTGGAACAAGATGAGGAAGAGGACGAGGAGCTGACCCTGAAGTACGG

CGCCAAGCACGTGATCATGCTCTTCGTGCCCGTGACTCTCTGCATGGTGGTGGTGGTGGCT

ACAATCAAGAGCGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCA

CAGAAGACACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATCCTGAATGCCGCCATCAT

GATCAGCGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTAT

AAGGTCATCCATGCCTGGCTGATCATATCATCTCTGTTGCTGCTGTTCTTTTTTAGCTTCA

TTTACCTGGGCGAAGTGTTTAAAACCTATAACGTTGCCGTGGACTACATTACTGTTGCCCT

CCTGATCTGGAACTTCGGCGTGGTGGGCATGATTTCCATTCACTGGAAAGGCCCCCTGAGA

CTGCAGCAGGCATACCTCATTATGATCTCCGCCCTCATGGCCCTGGTGTTCATCAAGTACC

TGCCCGAGTGGACTGCTTGGCTCATCTTGGCTGTGATCTCCGTGTATGATTTAGTGGCTGT

TCTGTGTCCTAAAGGTCCACTGCGTATGCTGGTGGAAACAGCTCAGGAAAGAAATGAAACA

CTGTTTCCTGCTCTGATTTACTCCTCAACAATGGTGTGGCTCGTGAATATGGCCGAAGGAG

ACCCTGAAGCCCAACGGAGAGTGTCCAAAAACTCCAAGTATAACGCCGAGAGCACAGAAAG

GGAGAGCCAGGATACAGTTGCCGAGAATGACGATGGCGGCTTCAGTGAGGAATGGGAAGCC

CAGAGGGACAGCCACCTGGGGCCTCACAGAAGCACCCCTGAGTCTAGAGCCGCTGTCCAGG

AACTGTCCAGCTCCATCCTGGCCGGCGAAGACCCCGAAGAAAGGGGAGTAAAACTTGGACT

GGGAGATTTCATCTTCTACAGTGTTCTCGTTGGCAAAGCCAGCGCAACAGCTAGCGGAGAC

TGGAACACAACAATAGCCTGTTTCGTAGCCATCTTAATTGGCCTGTGCCTTACACTTCTGC

TCCTGGCCATCTTCAAGAAGGCCCTGCCAGCCCTGCCTATCAGCATCACCTTCGGGCTTGT

TTTCTACTTTGCCACCGATTATCTGGTGCAGCCCTTCATGGACCAGCTGGCCTTCCACCAG

TTTTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACC

CCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCT

AATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGG

AGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCT

ATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTG

GGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACC

AGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGG

TCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACA

GGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCG

TCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATG

TTCCAAATTCGAAATTTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCC

CCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCC

CATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGGTGA

ATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAATAGGCAGAAATCGAGCCTCAA

AGAGCTTACAGTCTGGTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAG

GAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAG

GGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC

TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT

GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGT

ATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTAC

GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTA

CACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT

CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT

TTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGC

CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT

GTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATT

TTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT

TTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGC

CGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGT

CTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA

GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTT

ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAAT

GTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA

GACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAG

ACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCG

CTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGC

CGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC

GGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG

TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGG

CGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC

ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACC

AAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGA

TGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCG

AGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCA

TGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCG

CTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATC

GCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAG

ACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG

GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG

GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA

TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT

GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAA

AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT

CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT

TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTG

CCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

CAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC

GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG

TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA

CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT

ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG

GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC

GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC

TTTTGCTGGCCTTTTGCTCACATGT

(pAT053)

SEQ ID NO: 72

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGAGTGTAAACACAAAGATATTAGTACAAAAT

ACGTGAGGTAGAAAGTAATAATTTCTTGGGTAGTTTGGAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGAGACTAGAAAAGCCTTTTGACTACGAGGTT

TACATTTCGTTCAAAATCGTTTTCTATAGTTCAGCTGCTTCTGAGCTGTTGGAGACTAGAA

AAGCCTTTTGACTACGAGGTTTACATTTCGTTCAAAATCGTTTTCTATAGTTCAGCTGCTT

CTTTTTTCCGGGACGCGTCAATTGAGATCTCCGACATTGATTATTGACTAGTTATTAATAG

TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTA

CGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGAC

GTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA

CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG

ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTT

TCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCT

GCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTA

ATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGG

GCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCT

CCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCG

CGGCGGGCGGGAGCAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACG

CTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGG

CCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTAT

AGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTAT

TTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTC

TTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCT

GCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAG

CAGCTACAATCCAGCTACCATTCTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTC

TGAGTCCAAGCTAGGCCCTTTTGCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCT

CCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCA

ACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCA

CCATGGCCTACCCATACGATGTTCCAGATTACGCTACAGAATTACCTGCCCCCTTGAGCTA

CTTCCAGAATGCACAGATGAGCGAGGACAACCACCTGAGCAATACTGTAGGTAGCCAGAAT

GACAACAGAGAACGGCAGGAACACAACGACAGGCGGAGCCTGGGCCACCCTGAGCCCCTGT

CTAATGGAAGACCCCAGGGTAACAGCAGACAGGTGGTGGAACAAGATGAGGAAGAGGACGA

GGAGCTGACCCTGAAGTACGGCGCCAAGCACGTGATCATGCTCTTCGTGCCCGTGACTCTC

TGCATGGTGGTGGTGGTGGCTACAATCAAGAGCGTCAGCTTTTATACCCGGAAGGATGGGC

AGCTAATCTATACCCCATTCACAGAAGACACCGAGACTGTGGGCCAGAGAGCCCTGCACTC

AATCCTGAATGCCGCCATCATGATCAGCGTCATTGTTGTCATGACTATCCTCCTGGTGGTT

CTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTGATCATATCATCTCTGTTGC

TGCTGTTCTTTTTTAGCTTCATTTACCTGGGCGAAGTGTTTAAAACCTATAACGTTGCCGT

GGACTACATTACTGTTGCCCTCCTGATCTGGAACTTCGGCGTGGTGGGCATGATTTCCATT

CACTGGAAAGGCCCCCTGAGACTGCAGCAGGCATACCTCATTATGATCTCCGCCCTCATGG

CCCTGGTGTTCATCAAGTACCTGCCCGAGTGGACTGCTTGGCTCATCTTGGCTGTGATCTC

CGTGTATGATTTAGTGGCTGTTCTGTGTCCTAAAGGTCCACTGCGTATGCTGGTGGAAACA

GCTCAGGAAAGAAATGAAACACTGTTTCCTGCTCTGATTTACTCCTCAACAATGGTGTGGC

TCGTGAATATGGCCGAAGGAGACCCTGAAGCCCAACGGAGAGTGTCCAAAAACTCCAAGTA

TAACGCCGAGAGCACAGAAAGGGAGAGCCAGGATACAGTTGCCGAGAATGACGATGGCGGC

TTCAGTGAGGAATGGGAAGCCCAGAGGGACAGCCACCTGGGGCCTCACAGAAGCACCCCTG

AGTCTAGAGCCGCTGTCCAGGAACTGTCCAGCTCCATCCTGGCCGGCGAAGACCCCGAAGA

AAGGGGAGTAAAACTTGGACTGGGAGATTTCATCTTCTACAGTGTTCTCGTTGGCAAAGCC

AGCGCAACAGCTAGCGGAGACTGGAACACAACAATAGCCTGTTTCGTAGCCATCTTAATTG

GCCTGTGCCTTACACTTCTGCTCCTGGCCATCTTCAAGAAGGCCCTGCCAGCCCTGCCTAT

CAGCATCACCTTCGGGCTTGTTTTCTACTTTGCCACCGATTATCTGGTGCAGCCCTTCATG

GACCAGCTGGCCTTCCACCAGTTTTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGA

GGGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCC

AGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCC

TTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAA

CCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCT

CACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTG

GGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTT

TCACCATATTGGCCAGGCTGGTCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCC

TCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCT

AGGGCTGTGCCAGCTGCCTCGTCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTA

GCTGTTTTCCTCATGAGAATGTTCCAAATTCGAAATTTCTATTTAACCATTATATATTTAC

TTGTTTGCTATTATCTCTGCCCCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTC

TTTTGCTTATCTAGATATGCCCATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAAC

AACTACTTGTGGAATTGGTGAATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAA

TAGGCAGAAATCCAGCCTCAAAGAGCTTACAGTCTGGTAAGAGGAATAAAATGTCTGCAAA

TAGCCACAGGACAGGTCAAAGGAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGA

AAGCACCCCAGACTTCCTTAGGGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGT

GATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAG

GTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCC

TGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAT

ACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG

TTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTT

CCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT

TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATG

GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCAC

GTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTAT

TCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTT

AACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCT

CAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCT

GACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCT

CCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGG

CCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCA

GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT

CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG

GAAGAGTCGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGAT

TGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACA

GACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTT

TTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTAT

CGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGG

AAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCT

CCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG

CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGA

AGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAA

CTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCG

ATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGG

CCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAA

GAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATT

CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACG

ATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTAGTTAGTCTAG

CTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG

CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT

CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA

CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC

ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAGTCATATATACTTTAGATTGATTTA

AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCA

AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG

ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG

CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTG

GCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCA

CTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCT

GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATA

AGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGAC

CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGG

AGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC

TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA

GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG

GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT

(pAT054)

SEQ ID NO: 73

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGAGGTAGAAAGTAATAATTTCTTGGGTAGTTTGGAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGAATTATGTTTCGAATTTGACTTTAGAGGTT

TACATTTCTAGTCAAAGAAGAAACATCCATTCAGCTGCTCCTGAGCTGTTGGAATTATGTT

TCGAATTTGACTTTAGAGGTTTACATTTCTAGTCAAAGAAGAAACATCCATTCAGCTGCTC

CTGAGCTGTTGGAATTATGTTTCGAATTTGACTTTAGAGGTTTACATTTCTAGTCAAAGAA

GAAACATCCATTCAGCTGCTCCTTTTTTCCGGGACGCGTCAATTGAGATCTCCGACATTGA

TTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG

AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG

CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA

CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA

TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCA

GTAGATGACCTTATGGGACTTTCCTACTTGGCAGTAGATCTACGTATTAGTCATCGCTATT

ACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAAT

TTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCG

CGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGC

AGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGG

CCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTCGTTTAGTGAACCGTCAGAT

CGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGC

CTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAG

AGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAAT

ATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAA

TGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGT

TAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAA

GAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTGTGGT

TGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCGTGTTCATACC

TCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTT

GGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGA

ATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGTTCCAGATTACGCTACAGA

ATTACCTGCCCCCTTGAGCTACTTCCAGAATGCACAGATGAGCGAGGACAACCACCTGAGC

AATACTGTACGTAGCCAGAATGACAACAGAGAACGGCAGGAACACAACGACAGGCGGAGCC

TGGGCCACCCTGAGCCCCTGTCTAATGGAAGACCCCAGGGTAACAGCAGACAGGTGGTGGA

ACAAGATGAGGAAGAGGACGAGGAGCTGACCCTGAAGTACGGCGCCAAGCACGTGATCATG

CTCTTCGTGCCCGTGACTCTCTGCATGGTGGTGGTGGTGGCTACAATCAAGAGCGTCAGCT

TTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGACACCGAGACTGT

GGGCCAGAGAGCCCTGCACTCAATCCTGAATGCCGCCATCATGATCAGCGTCATTGTTGTC

ATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGC

TGATCATATCATCTCTGTTGCTGCTGTTCTTTTTTAGCTTCATTTACCTGGGCGAAGTGTT

TAAAACCTATAACGTTGCCGTGGACTACATTACTGTTGCCCTCCTGATCTGGAACTTCGGC

GTGGTGGGCATGATTTCCATTCACTGGAAAGGCCCCCTGAGACTGCAGCAGGCATACCTCA

TTATGATCTCCGCCCTCATGGCCCTGGTGTTCATCAAGTACCTGCCCGAGTGGACTGCTTG

GCTCATCTTGGCTGTGATCTCCGTGTATGATTTAGTGGCTGTTCTGTGTCCTAAAGGTCCA

CTGCGTATGCTGGTGGAAACAGCTCAGGAAAGAAATGAAACACTGTTTCCTGCTCTGATTT

ACTCCTCAACAATGGTGTGGCTCGTGAATATGGCCGAAGGAGACCCTGAAGCCCAACGGAG

AGTGTCCAAAAACTCCAAGTATAACGCCGAGAGCACAGAAAGGGAGAGCCAGGATACAGTT

GCCGAGAATGACGATGGCGGCTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGCCACCTGG

GGCCTCACAGAAGCACCCCTGAGTCTAGAGCCGCTGTCCAGGAACTGTCCAGCTCCATCCT

GGCCGGCGAAGACCCCGAAGAAAGGGGAGTAAAACTTGGACTGGGAGATTTCATCTTCTAC

AGTGTTCTCGTTGGCAAAGCCAGCGCAACAGCTAGCGGAGACTGGAACACAACAATAGCCT

GTTTCGTAGCCATCTTAATTGGCCTGTGCCTTACACTTCTGCTCCTGGCCATCTTCAAGAA

GGCCCTGCCAGCCCTGCCTATCAGCATCACCTTCGGGCTTGTTTTCTACTTTGCCACCGAT

TATCTGGTGCAGCCCTTCATGGACCAGCTGGCCTTCCACCAGTTTTACATCTAGTAAGCGG

CCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCT

GGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATC

ATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCA

AGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAG

TGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTG

CCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGT

TTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCCCCTCCTAATCTCA

GGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCC

TTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCGTCCCGTCACCTTCTGGCTT

CTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATGTTCCAAATTCGAAATTTCT

ATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCCCCCAGTAGATTGTTAGCTC

CAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCCCATCTGCCTGGTACAATCT

CTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGGTGAATGCATGAATAGAAGAATG

AGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCTCAAAGAGCTTACAGTCTGGTAA

GAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAGGAAGGAGGGGCTATTTCCA

GCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAGGGATAACAGGGTAATGGCG

CGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC

ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGA

GCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCT

GTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCAT

TAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGC

GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAA

GCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCA

AAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCG

CCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACA

CTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATT

GGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTT

TACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCC

CGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT

ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC

GAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATA

ATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT

GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT

GCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAGACAGGATGAGGATCGTTTC

GCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATT

CGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCA

GCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGC

AAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCT

CGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT

CTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGC

GGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGA

GCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCAT

CAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGG

ATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTT

TTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTG

GCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT

ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTT

CTGAACGAGCGTGAGACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAA

CTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA

AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT

GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA

GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA

TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC

TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA

CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGC

TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA

CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGT

GTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTG

CTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT

CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA

GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA

AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG

GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA

TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC

ACATGT

(pAT055)

SEQ ID NO: 74

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGAGACTAGAAAAGCCTTTTGACTACGAGGTT

TACATTTCGTTCAAAATCGTTTTCTATAGTTCAGCTGCTTCTGAGCTGTTGGAGACTAGAA

AAGCCTTTTGACTACGAGGTTTACATTTCGTTCAAAATCGTTTTCTATAGTTCAGCTGCTT

CTGAGCTGTTGGAGACTAGAAAAGCCTTTTGACTACGAGGTTTACATTTCGTTCAAAATCG

TTTTCTATAGTTCAGCTGCTTCTTTTTTCCGGGACGCGTCAATTGAGATCTCCGACATTGA

TTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG

AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG

CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA

CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA

TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCA

GTAGATGACCTTATGGGACTTTCCTACTTGGGAGTAGATCTAGGTATTAGTCATCGCTATT

ACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAAT

TTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCG

CGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGC

AGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGG

CCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTCGTTTAGTGAACCGTCAGAT

CGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGC

CTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAG

AGTGACGTAAGTAGCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAAT

ATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAA

TGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGT

TAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAA

GAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTGTGGT

TGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCGTGTTCATACC

TCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTT

GGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGA

ATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGTTCCAGATTACGCTACAGA

ATTACCTGCCCCCTTGAGCTACTTCCAGAATGCACAGATGAGCGAGGACAACCACCTGAGC

AATACTGTACGTAGCCAGAATGACAACAGAGAACGGCAGGAACACAACGACAGGCGGAGCC

TGGGCCACCCTGAGCCCCTGTCTAATGGAAGACCCCAGGGTAACAGCAGACAGGTGGTGGA

ACAAGATGAGGAAGAGGACGAGGAGCTGACCCTGAAGTACGGCGCCAAGCACGTGATCATG

CTCTTCGTGCCCGTGACTCTCTGCATGGTGGTGGTGGTGGCTACAATCAAGAGCGTCAGCT

TTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGACACCGAGACTGT

GGGCCAGAGAGCCCTGCACTCAATCCTGAATGCCGCCATCATGATCAGCGTCATTGTTGTC

ATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGC

TGATCATATCATCTCTGTTGCTGCTGTTCTTTTTTAGCTTCATTTACCTGGGCGAAGTGTT

TAAAACCTATAACGTTGCCGTGGACTACATTACTGTTGCCCTCCTGATCTGGAACTTCGGC

GTGGTGGGCATGATTTCCATTCACTGGAAAGGCCCCCTGAGACTGCAGCAGGCATACCTCA

TTATGATCTCCGCCCTCATGGCCCTGGTGTTCATCAAGTACCTGCCCGAGTGGACTGCTTG

GCTCATCTTGGCTGTGATCTCCGTGTATGATTTAGTGGCTGTTCTGTGTCCTAAAGGTCCA

CTGCGTATGCTGGTGGAAACAGCTCAGGAAAGAAATGAAACACTGTTTCCTGCTCTGATTT

ACTCCTCAACAATGGTGTGGCTCGTGAATATGGCCGAAGGAGACCCTGAAGCCCAACGGAG

AGTGTCCAAAAACTCCAAGTATAACGCCGAGAGCACAGAAAGGGAGAGCCAGGATACAGTT

GCCGAGAATGACGATGGCGGCTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGCCACCTGG

GGCCTCACAGAAGCACCCCTGAGTCTAGAGCCGCTGTCCAGGAACTGTCCAGCTCCATCCT

GGCCGGCGAAGACCCCGAAGAAAGGGGAGTAAAACTTGGACTGGGAGATTTCATCTTCTAC

AGTGTTCTCGTTGGCAAAGCCAGCGCAACAGCTAGCGGAGACTGGAACACAACAATAGCCT

GTTTCGTAGCCATCTTAATTGGCCTGTGCCTTACACTTCTGCTCCTGGCCATCTTCAAGAA

GGCCCTGCCAGCCCTGCCTATCAGCATCACCTTCGGGCTTGTTTTCTACTTTGCCACCGAT

TATCTGGTGCAGCCCTTCATGGACCAGCTGGCCTTCCACCAGTTTTACATCTAGTAAGCGG

CCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCT

GGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATC

ATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCA

AGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAG

TGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTG

CCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGT

TTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCCCCTCCTAATCTCA

GGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCC

TTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCGTCCCGTCACCTTCTGGCTT

CTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATGTTCCAAATTCGAAATTTCT

ATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCCCCCAGTAGATTGTTAGCTC

CAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCCCATCTGCCTGGTACAATCT

CTGGCACATGTTACAGGCAACAACTAGTTGTGGAATTGGTGAATGCATGAATAGAAGAATG

AGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCTCAAAGAGCTTACAGTCTGGTAA

GAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAGGAAGGAGGGGCTATTTCCA

GCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAGGGATAACAGGGTAATGGCG

CGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC

ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGA

GCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCT

GTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCAT

TAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGC

GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAA

GCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCA

AAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCG

CCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACA

CTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATT

GGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTT

TACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCC

CGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT

ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC

GAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATA

ATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT

GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT

GCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAGACAGGATGAGGATCGTTTC

GCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATT

CGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCA

GCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGC

AAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCT

CGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT

CTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGC

GGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGA

GCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCAT

CAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGG

ATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTT

TTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTG

GCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT

ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTT

CTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAA

CTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA

AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT

GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA

GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA

TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC

TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA

CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGC

TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA

CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGT

GTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTG

CTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT

CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA

GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA

AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG

GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA

TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC

ACATGT

(pAT056)

SEQ ID NO: 75

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATCCGGGACGCGTCAATTGAGATCTCCGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACA

TAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA

TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA

GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTAT

GGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCC

ACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA

TTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG

GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCG

GCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCG

AAGCGCGCGGCGGGCGGGAGCAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCC

ATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGA

ATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTAC

CGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTT

ATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATC

ATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAA

TATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTG

CTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTGTGGTTGGGATAAGGCTGG

ATTATTCTGAGATATCGGTACCTGAGCTGTTGGAGACTAGAAAAGCCTTTTGACTACGAGG

TTTACATTTCGTTCAAAATCGTTTTCTATAGTTCAGCTGCTTCTGAGCTGTTGGAGACTAG

AAAAGCCTTTTGACTACGAGGTTTACATTTCGTTCAAAATCGTTTTCTATAGTTCAGCTGC

TTCTGAGCTGTTGGAGACTAGAAAAGCCTTTTGACTACGAGGTTTACATTTCGTTCAAAAT

CGTTTTCTATAGTTCAGCTGCTTCGTCGACGCTAGGCCCTTTTGCTAATCGTGTTCATACC

TCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTT

GGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGA

ATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGTTCCAGATTACGCTACAGA

ATTACCTGCCCCCTTGAGCTACTTCCAGAATGCACAGATGAGCGAGGACAACCACCTGAGC

AATACTGTACGTAGCCAGAATGACAACAGAGAACGGCAGGAACACAACGACAGGCGGAGCC

TGGGCCACCCTGAGCCCCTGTCTAATGGAAGACCCCAGGGTAACAGCAGACAGGTGGTGGA

ACAAGATGAGGAAGAGGACGAGGAGCTGACCCTGAAGTACGGCGCCAAGCACGTGATCATG

CTCTTCGTGCCCGTGACTCTCTGCATGGTGGTGGTGGTGGCTACAATCAAGAGCGTCAGCT

TTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGACACCGAGACTGT

GGGCCAGAGAGCCCTGCACTCAATCCTGAATGCCGCCATCATGATCAGCGTCATTGTTGTC

ATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGC

TGATCATATCATCTCTGTTGCTGCTGTTCTTTTTTAGCTTCATTTACCTGGGCGAAGTGTT

TAAAACCTATAACGTTGCCGTGGACTACATTACTGTTGCCCTCCTGATCTGGAACTTCGGC

GTGGTGGGCATGATTTCCATTCACTGGAAAGGCCCCCTGAGACTGCAGCAGGCATACCTCA

TTATGATCTCCGCCCTCATGGCCCTGGTGTTCATCAAGTACCTGCCCGAGTGGACTGCTTG

GCTCATCTTGGCTGTGATCTCCGTGTATGATTTAGTGGCTGTTCTGTGTCCTAAAGGTCCA

CTGCGTATGCTGGTGGAAACAGCTCAGGAAAGAAATGAAACACTGTTTCCTGCTCTGATTT

ACTCCTCAACAATGGTGTGGCTCGTGAATATGGCCGAAGGAGACCCTGAAGCCCAACGGAG

AGTGTCCAAAAACTCCAAGTATAACGCCGAGAGCACAGAAAGGGAGAGCCAGGATACAGTT

GCCGAGAATGACGATGGCGGCTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGCCACCTGG

GGCCTCACAGAAGCACCCCTGAGTCTAGAGCCGCTGTCCAGGAACTGTCCAGCTCCATCCT

GGCCGGCGAAGACCCCGAAGAAAGGGGAGTAAAACTTGGACTGGGAGATTTCATCTTCTAC

AGTGTTCTCGTTGGCAAAGCCAGCGCAACAGCTAGCGGAGACTGGAACACAACAATAGCCT

GTTTCGTAGCCATCTTAATTGGCCTGTGCCTTACACTTCTGCTCCTGGCCATCTTCAAGAA

GGCCCTGCCAGCCCTGCCTATCAGCATCACCTTCGGGCTTGTTTTCTACTTTGCCACCGAT

TATCTGGTGCAGCCCTTCATGGACCAGCTGGCCTTCCACCAGTTTTACATCTAGTAAGCGG

CCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCT

GGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATC

ATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCA

AGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAG

TGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTG

CCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGT

TTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCCCCTCCTAATCTCA

GGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCC

TTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCGTCCCGTCACCTTCTGGCTT

CTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATGTTCCAAATTCGAAATTTCT

ATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCCCCCAGTAGATTGTTAGCTC

CAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCCCATCTGCCTGGTACAATCT

CTGGCACATGTTACAGGCAACAACTAGTTGTGGAATTGGTGAATGCATGAATAGAAGAATG

AGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCTCAAAGAGCTTACAGTCTGGTAA

GAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAGGAAGGAGGGGCTATTTCCA

GCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAGGGATAACAGGGTAATGGCG

CGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC

ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGA

GCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCT

GTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCAT

TAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGC

GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAA

GCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCA

AAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCG

CCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACA

CTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATT

GGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTT

TACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCC

CGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTT

ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC

GAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATA

ATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT

GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT

GCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAGACAGGATGAGGATCGTTTC

GCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATT

CGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCA

GCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGC

AAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCT

CGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT

CTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGC

GGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGA

GCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCAT

CAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGG

ATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTT

TTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTG

GCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT

ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTT

CTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAA

CTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA

AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT

GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA

GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA

TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC

TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA

CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGC

TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA

CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGT

GTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTG

CTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT

CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA

GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA

AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG

GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA

TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC

ACATGT

(pAT057)

SEQ ID NO: 76

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGAGGTAGAAAGTAATAATTTCTTGGGTAGTTTGGAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGAAGCTTGGATGGTCTTGTATTCAGGAGGTT

TACATTTCCTATACAACTGCATCCAATGATTCAGCTGCTTCTTTTTCCGGGACGCGTCAAT

TGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAG

TTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTG

ACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA

ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG

TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC

CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC

GTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCC

CCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGG

GCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAG

GCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTCG

TTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGA

CACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACG

CGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAA

ATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTC

TTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAA

CAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATAT

AAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT

CTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTT

GCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGT

GTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTC

TCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGT

TCCAGATTACGCTCTCACATTCATGGCCTCTGACAGCGAGGAAGAAGTGTGTGATGAGCGG

ACGTCCCTAATGTCGGCTGAGAGCCCCACGCCGCGCTCCTGCCAGGAGGGCAGGCAGGGCC

CAGAGGATGGAGAGAACACTGCCCAGTGGAGAAGCCAGGAGAACGAGGAGGACGGTGAGGA

GGACCCTGACCGCTATGTCTGTAGTGGGGTTCCCGGGCGGCCGCCAGGCCTGGAGGAAGAG

CTGACCCTCAAATACGGAGCGAAGCACGTGATCATGCTGTTTGTGCCTGTCACTCTGTGCA

TGATCGTGGTGGTAGCCACCATCAAGTCTGTGCGCTTCTACACAGAGAAGAATGGACAGCT

CATCTACACGCCATTCACTGAGGACACACCCTCGGTGGGCCAGCGCCTCCTCAACTCCGTG

CTGAACACCCTCATCATGATCAGCGTCATCGTGGTTATGACCATCTTCTTGGTGGTGCTCT

ACAAGTACCGCTGCTACAAGTTCATCCATGGCTGGTTGATCATGTCTTCACTGATGCTGCT

GTTCCTCTTCACCTATATCTACCTTGGGGAAGTGCTCAAGACCTACAATGTGGCCATGGAC

TACCCCACCCTCTTGCTGACTGTCTGGAACTTCGGGGCAGTGGGCATGGTGTGCATCCACT

GGAAGGGCCCTCTGGTGCTGCAGCAGGCCTACCTCATCATGATCAGTGCGCTCATGGCCCT

AGTGTTCATCAAGTACCTCCCAGAGTGGTCCGCGTGGGTCATCCTGGGCGCCATCTCTGTG

TATGATCTCGTGGCTGTGCTGTGTCCCAAAGGGCCTCTGAGAATGCTGGTAGAAACTGCCC

AGGAGAGAAATGAGCCCATATTCCCTGCCCTGATATACTCATCTGCCATGGTGTGGACGGT

TGGCATGGCGAAGCTGGACCCCTCCTCTCAGGGTGCCCTCCAGCTCCCCTACGACCCGGAG

ATGGAAGAAGACTCCTATGACAGTTTTGGGGAGCCTTCATACCCCGAAGTCTTTGAGCCTC

CCTTGACTGGCTACCCAGGGGAGGAGCTGGAGGAAGAGGAGGAAAGGGGCGTGAAGCTTGG

CCTCGGGGACTTCATCTTCTACAGTGTGCTGGTGGGCAAGGCGGCTGCCACGGGCAGCGGG

GACTGGAATACCACGCTGGCCTGCTTCGTGGCCATCCTCATTGGCTTGTGTCTGACCCTCC

TGCTGCTTGCTGTGTTCAAGAAGGCGCTGCCCGCCCTCCCCATCTCCATCACGTTCGGGCT

CATCTTTTACTTCTCCACGGACAACCTGGTGCGGCCGTTCATGGACACCCTGGCCTCCCAT

CAGCTCTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTG

ACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGT

CCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGG

TGGAGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGG

TCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTC

CTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATG

ACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGC

TGGTCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATT

ACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCC

TCGTCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGA

ATGTTCCAAATTCGAAATTTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCT

GCCCCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATAT

GCCCATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGG

TGAATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCT

CAAAGAGCTTAGAGTCTGGTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGAGAGGTCA

AAGGAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCT

TAGGGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCC

CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGC

TTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGC

GGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAG

TACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG

CTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCAC

GTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGT

GCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCAT

CGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACT

CTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGG

ATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAAGGCGA

ATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT

TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTC

AGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATT

TTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGA

AATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA

TGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAA

GAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGG

CCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGA

TGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTG

TCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG

GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT

GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCC

ATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACC

ACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCA

GGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAG

GCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATA

TCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGA

TCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGG

GCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCT

ATCGCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCA

ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA

TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG

CTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA

CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA

CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA

ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT

AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT

TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT

TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT

TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGA

TACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC

ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT

GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA

CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT

CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT

GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG

CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG

GCCTTTTGCTGGCCTTTTGCTCACATGT

(pAT058)

SEQ ID NO: 77

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGATTCCAGTTGTTATGTTTTATCCAGAGGTT

TACATTTCTGTAAAACTATACAACTGCATTTCAGCTGCTTCTTTTTCCGGGACGCGTCAAT

TGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAG

TTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTG

ACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA

ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG

TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAGGGTAAATGGCC

CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC

GTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCC

CCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGG

GCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAG

GCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTCG

TTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGA

CACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACG

CGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAA

ATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTC

TTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAA

CAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATAT

AAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT

CTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTT

GCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGT

GTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTC

TCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGT

TCCAGATTACGCTCTCACATTCATGGCCTCTGACAGCGAGGAAGAAGTGTGTGATGAGCGG

ACGTCCCTAATGTCGGCTGAGAGCCCCACGCCGCGCTCCTGCCAGGAGGGCAGGCAGGGCC

CAGAGGATGGAGAGAACACTGCCCAGTGGAGAAGCCAGGAGAACGAGGAGGACGGTGAGGA

GGACCCTGACCGCTATGTCTGTAGTGGGGTTCCCGGGCGGCCGCCAGGCCTGGAGGAAGAG

CTGACCCTCAAATACGGAGCGAAGCACGTGATCATGCTGTTTGTGCCTGTCACTCTGTGCA

TGATCGTGGTGGTAGCCACCATCAAGTCTGTGCGCTTCTACACAGAGAAGAATGGACAGCT

CATCTACACGCCATTCACTGAGGACACACCCTCGGTGGGCCAGCGCCTCCTCAACTCCGTG

CTGAACACCCTCATCATGATCAGCGTCATCGTGGTTATGACCATCTTCTTGGTGGTGCTCT

ACAAGTACCGCTGCTACAAGTTCATCCATGGCTGGTTGATCATGTCTTCACTGATGCTGCT

GTTCCTCTTCACCTATATCTACCTTGGGGAAGTGCTCAAGACCTACAATGTGGCCATGGAC

TACCCCACCCTCTTGCTGACTGTCTGGAACTTCGGGGCAGTGGGCATGGTGTGCATCCACT

GGAAGGGCCCTCTGGTGCTGCAGCAGGCCTACCTCATCATGATCAGTGCGCTCATGGCCCT

AGTGTTCATCAAGTACCTCCCAGAGTGGTCCGCGTGGGTCATCCTGGGCGCCATCTCTGTG

TATGATCTCGTGGCTGTGCTGTGTCCCAAAGGGCCTCTGAGAATGCTGGTAGAAACTGCCC

AGGAGAGAAATGAGCCCATATTCCCTGCCCTGATATACTCATCTGCCATGGTGTGGACGGT

TGGCATGGCGAAGCTGGACCCCTCCTCTCAGGGTGCCCTCCAGCTCCCCTACGACCCGGAG

ATGGAAGAAGACTCCTATGACAGTTTTGGGGAGCCTTCATACCCCGAAGTCTTTGAGCCTC

CCTTGACTGGCTACCCAGGGGAGGAGCTGGAGGAAGAGGAGGAAAGGGGCGTGAAGCTTGG

CCTCGGGGACTTCATCTTCTACAGTGTGCTGGTGGGCAAGGCGGCTGCCACGGGCAGCGGG

GACTGGAATACCACGCTGGCCTGCTTCGTGGCCATCCTCATTGGCTTGTGTCTGACCCTCC

TGCTGCTTGCTGTGTTCAAGAAGGCGCTGCCCGCCCTCCCCATCTCCATCACGTTCGGGCT

CATCTTTTACTTCTCCACGGACAACCTGGTGCGGCCGTTCATGGACACCCTGGCCTCCCAT

CAGCTCTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTG

ACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGT

CCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGG

TGGAGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGG

TCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTC

CTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATG

ACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGC

TGGTCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATT

ACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCC

TCGTCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGA

ATGTTCCAAATTCGAAATTTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCT

GCCCCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATAT

GCCCATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGG

TGAATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCT

CAAAGAGCTTAGAGTCTGGTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGAGAGGTCA

AAGGAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCT

TAGGGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCC

CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGC

TTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGC

GGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAG

TACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG

CTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCAC

GTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGT

GCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCAT

CGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACT

CTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGG

ATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAAGGCGA

ATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT

TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTC

AGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATT

TTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGA

AATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA

TGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAA

GAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGG

CCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGA

TGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTG

TCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG

GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT

GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCC

ATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACC

ACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCA

GGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAG

GCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATA

TCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGA

TCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGG

GCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCT

ATCGCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCA

ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA

TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG

CTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA

CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA

CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA

ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT

AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT

TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT

TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT

TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGA

TACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC

ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT

GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA

CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT

CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT

GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG

CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG

GCCTTTTGCTGGCCTTTTGCTCACATGT

(pAT059)

SEQ ID NO: 78

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGATTCGCTTAATTCGAATATCCTAAGAGGTT

TACATTTCTTGATATTACTATTAAGCCACTTCAGCTGCTTCTTTTTCCGGGACGCGTCAAT

TGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAG

TTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTG

ACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA

ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG

TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC

CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC

GTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCC

CCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGG

GCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAG

GCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTCG

TTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGA

CACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACG

CGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAA

ATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTC

TTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAA

CAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATAT

AAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT

CTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTT

GCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGT

GTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTC

TCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGT

TCCAGATTACGCTCTCACATTCATGGCCTCTGACAGCGAGGAAGAAGTGTGTGATGAGCGG

ACGTCCCTAATGTCGGCTGAGAGCCCCACGCCGCGCTCCTGCCAGGAGGGCAGGCAGGGCC

CAGAGGATGGAGAGAACACTGCCCAGTGGAGAAGCCAGGAGAACGAGGAGGAGGGTGAGGA

GGACCCTGACCGCTATGTCTGTAGTGGGGTTCCCGGGCGGCCGCCAGGCCTGGAGGAAGAG

CTGACCCTCAAATACGGAGCGAAGCACGTGATCATGCTGTTTGTGCCTGTCACTCTGTGCA

TGATCGTGGTGGTAGCCACCATCAAGTCTGTGCGCTTCTACACAGAGAAGAATGGACAGCT

CATCTACACGCCATTCACTGAGGACACACCCTCGGTGGGCCAGCGCCTCCTCAACTCCGTG

CTGAACACCCTCATCATGATCAGCGTCATCGTGGTTATGACCATCTTCTTGGTGGTGCTCT

ACAAGTACCGCTGCTACAAGTTCATCCATGGCTGGTTGATCATGTCTTCACTGATGCTGCT

GTTCCTCTTCACCTATATCTACCTTGGGGAAGTGCTCAAGACCTACAATGTGGCCATGGAC

TACCCCACCCTCTTGCTGACTGTCTGGAACTTCGGGGCAGTGGGCATGGTGTGCATCCACT

GGAAGGGCCCTCTGGTGCTGCAGCAGGCCTACCTCATCATGATCAGTGCGCTCATGGCCCT

AGTGTTCATCAAGTACCTCCCAGAGTGGTCCGCGTGGGTCATCCTGGGCGCCATCTCTGTG

TATGATCTCGTGGCTGTGCTGTGTCCCAAAGGGCCTCTGAGAATGCTGGTAGAAACTGCCC

AGGAGAGAAATGAGCCCATATTCCCTGCCCTGATATACTCATCTGCCATGGTGTGGACGGT

TGGCATGGCGAAGCTGGACCCCTCCTCTCAGGGTGCCCTCCAGCTCCCCTACGACCCGGAG

ATGGAAGAAGACTCCTATGACAGTTTTGGGGAGCCTTCATACCCCGAAGTCTTTGAGCCTC

CCTTGACTGGCTACCCAGGGGAGGAGCTGGAGGAAGAGGAGGAAAGGGGCGTGAAGCTTGG

CCTCGGGGACTTCATCTTCTACAGTGTGCTGGTGGGCAAGGCGGCTGCCACGGGCAGCGGG

GACTGGAATACCACGCTGGCCTGCTTCGTGGCCATCCTCATTGGCTTGTGTCTGACCCTCC

TGCTGCTTGCTGTGTTCAAGAAGGCGCTGCCCGCCCTCCCCATCTCCATCACGTTCGGGCT

CATCTTTTACTTCTCCACGGACAACCTGGTGCGGCCGTTCATGGACACCCTGGCCTCCCAT

CAGCTCTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTG

ACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGT

CCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGG

TGGAGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGG

TCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTC

CTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATG

ACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGC

TGGTCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATT

ACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCC

TCGTCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGA

ATGTTCCAAATTCGAAATTTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCT

GCCCCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATAT

GCCCATCTGCCTGGTACAATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGG

TGAATGCATGAATAGAAGAATGAGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCT

CAAAGAGCTTACAGTCTGGTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCA

AAGGAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCT

TAGGGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCC

CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGC

TTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGC

GGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAG

TACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG

CTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCAC

GTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGT

GCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCAT

CGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACT

CTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGG

ATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGA

ATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGA

TGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT

TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTC

AGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATT

TTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGA

AATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA

TGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAA

GAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGG

CCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGA

TGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTG

TCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG

GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT

GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCC

ATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACC

ACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCA

GGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAG

GCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATA

TCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGA

TCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGG

GCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCT

ATCGCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCA

ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA

TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG

CTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA

CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA

CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA

ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT

AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT

TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT

TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT

TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGA

TACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC

ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG

TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT

GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA

CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT

CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT

GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG

CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG

GCCTTTTGCTGGCCTTTTGCTCACATGT

(pAT060)

SEQ ID NO: 79

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGAGTGTAAACACAAAGATATTAGTACAAAAT

ACGTGAGGTAGAAAGTAATAATTTCTTGGGTAGTTTGGAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGATTCGCTTAATTCGAATATCCTAAGAGGTT

TACATTTCTTGATATTACTATTAAGCCACTTCAGCTGCTTCTGAGCTGTTGGATTCGCTTA

ATTCGAATATCCTAAGAGGTTTACATTTCTTGATATTAGTATTAAGCCACTTCAGCTGCTT

CTTTTTCCGGGACGCGTCAATTGAGATCTCCGACATTGATTATTGACTAGTTATTAATAGT

AATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTAC

GGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACG

TATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTAC

GGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGA

CGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT

CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTG

CTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAA

TTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG

CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTC

CGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGC

GGCGGGCGGGAGCAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGC

TGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGC

CGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATA

GAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATT

TCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCT

TTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTG

CATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGC

AGCTACAATCCAGCTACCATTCTGCTTTTATTTTGTGGTTGGGATAAGGCTGGATTATTCT

GAGTCCAAGCTAGGCCCTTTTGCTAATCGTGTTCATACCTCTTATCTTCCTCCCACAGCTC

CTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTACCGGTGGCAA

CGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCACGCCCCAGAGCCGCCAC

CATGGCCTACCCATACGATGTTCCAGATTACGCTCTCACATTCATGGCCTCTGACAGCGAG

GAAGAAGTGTGTGATGAGCGGACGTCCCTAATGTCGGCTGAGAGCCCCACGCCGCGCTCCT

GCCAGGAGGGCAGGCAGGGCCCAGAGGATGGAGAGAACACTGCCCAGTGGAGAAGCCAGGA

GAACGAGGAGGACGGTGAGGAGGACCCTGACCGCTATGTCTGTAGTGGGGTTCCCGGGCGG

CCGCCAGGCCTGGAGGAAGAGCTGACCCTCAAATACGGAGCGAAGCACGTGATCATGCTGT

TTGTGCCTGTCACTCTGTGCATGATCGTGGTGGTAGCCACCATCAAGTCTGTGCGCTTCTA

CACAGAGAAGAATGGACAGCTCATCTACACGCCATTCACTGAGGACACACCCTCGGTGGGC

CAGCGCCTCCTCAACTCCGTGCTGAACACCCTCATCATGATCAGCGTCATCGTGGTTATGA

CCATCTTCTTGGTGGTGCTCTACAAGTACCGCTGCTACAAGTTCATCCATGGCTGGTTGAT

CATGTCTTCACTGATGCTGCTGTTCCTCTTCACCTATATCTACCTTGGGGAAGTGCTCAAG

ACCTACAATGTGGCCATGGACTACCCCACCCTCTTGCTGACTGTCTGGAACTTCGGGGCAG

TGGGCATGGTGTGCATCCACTGGAAGGGCCCTCTGGTGCTGCAGCAGGCCTACCTCATCAT

GATCAGTGCGCTCATGGCCCTAGTGTTCATCAAGTACCTCCCAGAGTGGTCCGCGTGGGTC

ATCCTGGGCGCCATCTCTGTGTATGATCTCGTGGCTGTGCTGTGTCCCAAAGGGCCTCTGA

GAATGCTGGTAGAAACTGCCCAGGAGAGAAATGAGCCCATATTCCCTGCCCTGATATACTC

ATCTGCCATGGTGTGGACGGTTGGCATGGCGAAGCTGGACCCCTCCTCTCAGGGTGCCCTC

CAGCTCCCCTACGACCCGGAGATGGAAGAAGACTCCTATGACAGTTTTGGGGAGCCTTCAT

ACCCCGAAGTCTTTGAGCCTCCCTTGACTGGCTACCCAGGGGAGGAGCTGGAGGAAGAGGA

GGAAAGGGGCGTGAAGCTTGGCCTCGGGGACTTCATCTTCTACAGTGTGCTGGTGGGCAAG

GCGGCTGCCACGGGCAGCGGGGACTGGAATACCACGCTGGCCTGCTTCGTGGCCATCCTCA

TTGGCTTGTGTCTGACCCTCCTGCTGCTTGCTGTGTTCAAGAAGGCGCTGCCCGCCCTCCC

CATCTCCATCACGTTCGGGCTCATCTTTTACTTCTCCACGGACAACCTGGTGCGGCCGTTC

ATGGACACCCTGGCCTCCCATCAGCTCTACATCTAGTAAGCGGCCGCCCTAGGGAGCTCCT

CGAGGGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCAC

TCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTG

TCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGGGGGGAAGA

CAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTG

GCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTG

TTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGG

GTTTCACCATATTGGCCAGGCTGGTCTCCCCCTCCTAATCTCAGGTGATCTACCCACCTTG

GCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCCTGGG

CCTAGGGCTGTGCCAGCTGCCTCGTCCCGTCACCTTCTGGCTTCTTCTCTCCCTCCATATC

TTAGCTGTTTTCCTCATGAGAATGTTCCAAATTCGAAATTTCTATTTAACCATTATATATT

TACTTGTTTGCTATTATCTCTGCCCCCAGTAGATTGTTAGCTCCAGAAGAGAAAGGATCAT

GTCTTTTGCTTATCTAGATATGCCCATCTGCCTGGTACAATCTCTGGCACATGTTACAGGC

AACAACTAGTTGTGGAATTGGTGAATGCATGAATAGAAGAATGAGTGAATGAATGAATAGA

CAATAGGCAGAAATCCAGGCTCAAAGAGCTTACAGTCTGGTAAGAGGAATAAAATGTCTGC

AAATAGCCACAGGACAGGTCAAAGGAAGGAGGGGCTATTTCCAGCTGAGGGCACCCCATCA

GGAAAGCACCCCAGACTTCCTTAGGGATAACAGGGTAATGGCGCGGGCCGCAGGAACCCCT

AGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCA

AAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCT

GCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCG

CATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG

TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTT

CTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTC

CCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTG

ATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTC

CACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGGC

TATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGA

TTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCAC

TCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCC

GCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCG

TCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAA

GGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACG

TCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATAC

ATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAA

AAGGAAGAGTCGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATG

GATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACA

ACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTT

CTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGC

TATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC

GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTT

GCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATC

CGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGAT

GGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCC

GAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATG

GCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTG

TGGCCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCT

GAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCG

ATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAACGAGCGTGACACC

ACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTC

TAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCT

GCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGG

TCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT

ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGC

CTGAGTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAGTCATATATACTTTAGATTGAT

TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGA

CCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA

AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA

CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA

CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCA

CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG

GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG

ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC

GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA

GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGG

AGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT

TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC

GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT

(pAT061)

SEQ ID NO: 80

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAA

GGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAAT

ACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAA

TGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

GTGGAAAGGACGAGGTACCGTGAGCTGTTGGATTCGCTTAATTCGAATATCCTAAGAGGTT

TACATTTCTTGATATTACTATTAAGCCACTTCAGCTGCTTCTGAGCTGTTGGATTCGCTTA

ATTCGAATATCCTAAGAGGTTTACATTTCTTGATATTAGTATTAAGCCACTTCAGCTGCTT

CTGAGCTGTTGGATTCGCTTAATTCGAATATCCTAAGAGGTTTACATTTCTTGATATTACT

ATTAAGCCACTTCAGCTGCTTCTTTTTCCGGGACGCGTCAATTGAGATCTCCGACATTGAT

TATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGA

GTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC

CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGAC

GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT

GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAG

TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA

CCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATT

TTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGC

GCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCA

GCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGC

CCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTCGTTTAGTGAACCGTCAGATC

GCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCC

TCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGA

GTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATA

TACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAAT

GATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTT

AAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAG

AGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTGTGGTT

GGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCGTGTTCATACCT

CTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTG

GCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAA

TTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGTTCCAGATTACGCTCTCACA

TTCATGGCCTCTGACAGCGAGGAAGAAGTGTGTGATGAGCGGACGTCCCTAATGTCGGCTG

AGAGCCCCACGCCGCGCTCCTGCCAGGAGGGCAGGCAGGGCCCAGAGGATGGAGAGAACAC

TGCCCAGTGGAGAAGCCAGGAGAACGAGGAGGACGGTGAGGAGGACCCTGACCGCTATGTC

TGTAGTGGGGTTCCCGGGCGGCCGCCAGGCCTGGAGGAAGAGCTGACCCTCAAATACGGAG

CGAAGCACGTGATCATGCTGTTTGTGCCTGTCACTCTGTGCATGATCGTGGTGGTAGCCAC

CATCAAGTCTGTGCGCTTCTACACAGAGAAGAATGGACAGCTCATCTACACGCCATTCACT

GAGGACACACCCTCGGTGGGCCAGCGCCTCCTCAACTCCGTGCTGAACACCCTCATCATGA

TCAGCGTCATCGTGGTTATGACCATCTTCTTGGTGGTGCTCTACAAGTACCGCTGCTACAA

GTTCATCCATGGCTGGTTGATCATGTCTTCACTGATGCTGCTGTTCCTCTTCACCTATATC

TACCTTGGGGAAGTGCTCAAGACCTACAATGTGGCCATGGACTACCCCACCCTCTTGCTGA

CTGTCTGGAACTTCGGGGCAGTGGGCATGGTGTGCATCCACTGGAAGGGCCCTCTGGTGCT

GCAGCAGGCCTACCTCATCATGATCAGTGCGCTCATGGCCCTAGTGTTCATCAAGTACCTC

CCAGAGTGGTCCGCGTGGGTCATCCTGGGCGCCATCTCTGTGTATGATCTCGTGGCTGTGC

TGTGTCCCAAAGGGCCTCTGAGAATGCTGGTAGAAACTGCCCAGGAGAGAAATGAGCCCAT

ATTCCCTGCCCTGATATACTCATCTGCCATGGTGTGGACGGTTGGCATGGCGAAGCTGGAC

CCCTCCTCTCAGGGTGCCCTCCAGCTCCCCTACGACCCGGAGATGGAAGAAGACTCCTATG

ACAGTTTTGGGGAGCCTTCATACCCCGAAGTCTTTGAGCCTCCCTTGACTGGCTACCCAGG

GGAGGAGCTGGAGGAAGAGGAGGAAAGGGGCGTGAAGCTTGGCCTCGGGGACTTCATCTTC

TACAGTGTGCTGGTGGGCAAGGCGGCTGCCACGGGCAGCGGGGACTGGAATACCACGCTGG

CCTGCTTCGTGGCCATCCTCATTGGCTTGTGTCTGACCCTCCTGCTGCTTGCTGTGTTCAA

GAAGGCGCTGCCCGCCCTCCCCATCTCCATCACGTTCGGGCTCATCTTTTACTTCTCCACG

GACAACCTGGTGCGGCCGTTCATGGACACCCTGGCCTCCCATCAGCTCTACATCTAGTAAG

CGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCT

CCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGC

ATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGA

GCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTG

GAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTC

CTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTT

TGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCCCCTCCTAATC

TCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCT

CCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCGTCCCGTCACCTTCTGG

CTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATGTTCCAAATTCGAAATT

TCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCCCCCAGTAGATTGTTAG

CTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCCCATCTGCCTGGTACAA

TCTCTGGCACATGTTACAGGCAACAACTAGTTGTGGAATTGGTGAATGCATGAATAGAAGA

ATGAGTGAATGAATGAATAGACAATAGGCAGAAATCGAGCCTCAAAGAGCTTACAGTCTGG

TAAGAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAGGAAGGAGGGGCTATTT

CCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAGGGATAACAGGGTAATG

GCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG

CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG

TGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCA

TCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCG

CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTT

AGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGT

CAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACC

CCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTT

TCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACA

ACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCT

ATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAAC

GTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAG

CCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCG

CTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATC

ACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATG

ATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTA

TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA

AATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAGACAGGATGAGGATCGT

TTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCT

ATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTG

TCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAAC

TGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGT

GCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAG

GATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGC

GGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCAT

CGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAG

CATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCG

AGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCG

CTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGCG

TTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGC

TTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTT

CTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTAT

TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGA

TAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA

TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC

CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG

ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC

TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGA

TCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTC

AGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC

TGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC

CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCT

AGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT

CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG

ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCAC

ACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAGAGCGTGAGCTATGA

GAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCG

GAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGT

CGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGC

CTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG

CTCACATGT

(pAT062)

SEQ ID NO: 81

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG

GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAC

TCCATCACTAGGGGTTCCTGCGGCCAATTCAGTGGATCCCGATAACTATAACGGTCCTAAG

GTAGCGATTTAAATCCGGGACGCGTCAATTGAGATCTCCGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACA

TAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA

TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA

GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTAT

GGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCC

ACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA

TTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG

GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCG

GCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCG

AAGCGCGCGGCGGGCGGGAGCAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCC

ATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGA

ATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTAC

CGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTT

ATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATC

ATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAA

TATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTG

CTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTGTGGTTGGGATAAGGCTGG

ATTATTCTGAGATATCGGTACCTGAGCTGTTGGATTCGCTTAATTCGAATATCCTAAGAGG

TTTACATTTCTTGATATTACTATTAAGCCACTTCAGCTGCTTCTGAGCTGTTGGATTCGCT

TAATTCGAATATCCTAAGAGGTTTACATTTCTTGATATTACTATTAAGCCACTTCAGCTGC

TTCTGAGCTGTTGGATTCGCTTAATTCGAATATCCTAAGAGGTTTACATTTCTTGATATTA

CTATTAAGCCACTTCAGCTGCTTCGTCGACGCTAGGCCCTTTTGCTAATCGTGTTCATACC

TCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTT

GGCAAAGAATTACCGGTGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGA

ATTCACGCCCCAGAGCCGCCACCATGGCCTACCCATACGATGTTCCAGATTACGCTCTCAC

ATTCATGGCCTCTGACAGCGAGGAAGAAGTGTGTGATGAGCGGACGTCCCTAATGTCGGCT

GAGAGCCCCACGCCGCGCTCCTGCCAGGAGGGCAGGCAGGGCCCAGAGGATGGAGAGAACA

CTGCCCAGTGGAGAAGCCAGGAGAACGAGGAGGACGGTGAGGAGGACCCTGACCGCTATGT

CTGTAGTGGGGTTCCCGGGCGGCCGCCAGGCCTGGAGGAAGAGCTGACCCTCAAATACGGA

GCGAAGCACGTGATCATGCTGTTTGTGCCTGTCACTCTGTGCATGATCGTGGTGGTAGCCA

CCATCAAGTCTGTGCGCTTCTACACAGAGAAGAATGGACAGCTCATCTACACGCCATTCAC

TGAGGACACACCCTCGGTGGGCCAGCGCCTCCTCAACTCCGTGCTGAACACCCTCATCATG

ATCAGCGTCATCGTGGTTATGACCATCTTCTTGGTGGTGCTCTACAAGTACCGCTGCTACA

AGTTCATCCATGGCTGGTTGATCATGTCTTCACTGATGCTGCTGTTCCTCTTCACCTATAT

CTACCTTGGGGAAGTGCTCAAGACCTACAATGTGGCCATGGACTACCCCACCCTCTTGCTG

ACTGTCTGGAACTTCGGGGCAGTGGGCATGGTGTGCATCCACTGGAAGGGCCCTCTGGTGC

TGCAGCAGGCCTACCTCATCATGATCAGTGCGCTCATGGCCCTAGTGTTCATCAAGTACCT

CCCAGAGTGGTCCGCGTGGGTCATCCTGGGCGCCATCTCTGTGTATGATCTCGTGGCTGTG

CTGTGTCCCAAAGGGCCTCTGAGAATGCTGGTAGAAACTGCCCAGGAGAGAAATGAGCCCA

TATTCCCTGCCCTGATATACTCATCTGCCATGGTGTGGACGGTTGGCATGGCGAAGCTGGA

CCCCTCCTCTCAGGGTGCCCTCCAGCTCCCCTACGACCCGGAGATGGAAGAAGACTCCTAT

GACAGTTTTGGGGAGCCTTCATACCCCGAAGTCTTTGAGCCTCCCTTGACTGGCTACCCAG

GGGAGGAGCTGGAGGAAGAGGAGGAAAGGGGCGTGAAGCTTGGCCTCGGGGACTTCATCTT

CTACAGTGTGCTGGTGGGCAAGGCGGCTGCCACGGGCAGCGGGGACTGGAATACCACGCTG

GCCTGCTTCGTGGCCATCCTCATTGGCTTGTGTCTGACCCTCCTGCTGCTTGCTGTGTTCA

AGAAGGCGCTGCCCGCCCTCCCCATCTCCATCACGTTCGGGCTCATCTTTTACTTCTCCAC

GGACAACCTGGTGCGGCCGTTCATGGACACCCTGGCCTCCCATCAGCTCTACATCTAGTAA

GCGGCCGCCCTAGGGAGCTCCTCGAGGGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTC

TCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTG

CATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGG

AGCAAGGGGCAAGGGGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCT

GGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCT

CCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTT

TTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCCCCTCCTAAT

CTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGC

TCCCTTCCCTGTCCTTCCTGGGCCTAGGGCTGTGCCAGCTGCCTCGTCCCGTCACCTTCTG

GCTTCTTCTCTCCCTCCATATCTTAGCTGTTTTCCTCATGAGAATGTTCCAAATTCGAAAT

TTCTATTTAACCATTATATATTTACTTGTTTGCTATTATCTCTGCCCCCAGTAGATTGTTA

GCTCCAGAAGAGAAAGGATCATGTCTTTTGCTTATCTAGATATGCCCATCTGCCTGGTACA

ATCTCTGGCACATGTTACAGGCAACAACTACTTGTGGAATTGGTGAATGCATGAATAGAAG

AATGAGTGAATGAATGAATAGACAATAGGCAGAAATCCAGCCTCAAAGAGCTTACAGTCTG

GTAAGAGGAATAAAATGTCTGCAAATAGCCACAGGACAGGTCAAAGGAAGGAGGGGCTATT

TCCAGCTGAGGGCACCCCATCAGGAAAGCACCCCAGACTTCCTTAGGGATAACAGGGTAAT

GGCGCGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTC

GCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCA

GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGC

GCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCT

TAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG

TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGAC

CCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT

TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAAC

AACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTC

TATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAA

CGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCA

GCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCC

GCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCAT

CACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCAT

GATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCT

ATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT

AAATGCTTCAATAATATTGAAAAAGGAAGAGTCGATCGATCAAGAGACAGGATGAGGATCG

TTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGC

TATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCT

GTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAA

CTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTG

TGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCA

GGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATG

CGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCA

TCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGA

GCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGC

GAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCC

GCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGC

GTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTG

CTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGT

TCTTCTGAACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTA

TTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGG

ATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAA

ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAG

CCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATA

GACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTA

CTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAG

ATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGT

CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTG

CTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA

CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTC

TAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGC

TCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG

GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCA

CACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG

AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC

GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG

TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG

CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT

GCTCACATGT

(endogenous PSEN1 specific forward primer)

SEQ ID NO: 82

CCTGACCACCTTGCACTATT

(endogenous PSEN1 specific probe)

SEQ ID NO: 83

TGTGTCCCTCGGTGCAGAAACTAC

(endogenous PSEN1 specific reverse primer)

SEQ ID NO: 84

CAACTTCCGGGCCTATCATATC

(plasmid-encoded transcript specific forward primer)

SEQ ID NO: 85

TGGACCAATTAGCATTCCATCA

(plasmid-encoded transcript specific probe)

SEQ ID NO: 86

TGAACTACGCCTGAGGATCCGATCT

(plasmid-encoded transcript specific reverse primer)

SEQ ID NO: 87

GCCAGAAGTCAGATGCTCAA

(endogenous PSEN2 specific forward primer)

SEQ ID NO: 88

GAGAAGGTCAGATTAGGGCG

(endogenous PSEN2 specific probe)

SEQ ID NO: 89

AAAGAGTGTGCTCGGGAGTGC

(endogenous PSEN2 specific reverse primer)

SEQ ID NO: 90

TCGTAGGGAACTGGCTTTTC

Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, that have been made throughout this disclosure are hereby incorporated herein by reference in their entirety for all purposes.

Although the present invention has been described with reference to specific details of certain embodiments thereof in the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

GENE THERAPY FOR NEURODEGENERATIVE DISORDERS USING POLYNUCLEOTIDE SILENCING AND REPLACEMENT

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATION

PCT Information

Provisional Applications (1)