阅读说明：本技术 用于增强基因表达的组合物和方法 (Compositions and methods for enhancing gene expression ) 是由 安娜希塔·喀拉瓦拉 于 2018-03-16 设计创作，主要内容包括：本公开提供了用于在哺乳动物细胞中表达基因的多核苷酸盒、表达载体和方法。(The present disclosure provides polynucleotide cassettes, expression vectors and methods for expressing genes in mammalian cells.)

1. A non-naturally occurring polynucleotide cassette for enhancing transgene expression in a mammalian cell, said non-naturally occurring polynucleotide cassette comprising, in 5 'to 3' order:

(a) A first enhancement sub-region;

(b) A promoter region;

(c) A coding sequence encoding a secreted polypeptide;

(d) A second enhancement sub-region; and

(e) A polyadenylation site;

Wherein the coding sequence is operably linked to the promoter region,

Wherein expression of the secreted polypeptide by the polynucleotide cassette in the mammalian cell is at least 5-fold greater than expression of the secreted polypeptide by the reference cassette in the mammalian cell, and

Wherein the reference cassette comprises, in 5 'to 3' order, a CMV enhancer sequence (SEQ ID NO:2), a CMV promoter (SEQ ID NO:21), a chimeric intron (SEQ ID NO:22), a 5'UTR (SEQ ID NO:23), a coding sequence encoding the secreted polypeptide, a 3' UTR (SEQ ID NO:25), and an SV40polyA sequence (SEQ ID NO: 26).

2. the polynucleotide cassette of claim 1, wherein the cassette is free of RNA export signals.

3. The polynucleotide cassette of any one of the preceding claims, wherein the first enhancer region comprises the Cytomegalovirus (CMV) sequence set forth in SEQ ID No. 1 or a sequence having at least 85% identity thereto.

4. The polynucleotide cassette of any one of the preceding claims, wherein the promoter region comprises the CMV promoter sequence shown in SEQ ID No. 4 or a sequence having at least 85% identity thereto.

5. The polynucleotide cassette of any one of the preceding claims, further comprising a non-translated region (5' UTR), said 5' UTR being downstream of said promoter region and upstream of said coding sequence, wherein said 5' UTR comprises in 5' to 3' order the TPL sequence according to SEQ ID No. 11 or a sequence having at least 85% identity thereto and the eMLP sequence according to SEQ ID No. 12 or a sequence having at least 85% identity thereto.

6. The polynucleotide cassette of any one of the preceding claims, wherein the second enhancer comprises the complete EES sequence shown in SEQ ID No. 13 or a sequence having at least 85% identity thereto.

7. The polynucleotide cassette of any one of the preceding claims, wherein the polyadenylation site comprises the Human Growth Hormone (HGH) polyadenylation site set forth in SEQ ID No. 14 or a sequence having at least 85% identity thereto.

8. The polynucleotide cassette of any one of the preceding claims, further comprising one or more regions comprising a sequence selected from SEQ ID NOs 76-80 or a sequence having at least 85% identity thereto.

9. the polynucleotide cassette of any one of the preceding claims, wherein the secreted polypeptide is an anti-angiogenic polypeptide.

10. The polynucleotide cassette of any one of the preceding claims, wherein the secreted polypeptide comprises soluble fms-like tyrosine kinase-1 (sFLT-1) or a VEGF-binding fragment of sFLT-1.

11. The polynucleotide cassette of claim 1, comprising 5 'to 3':

(a) A first enhancer region comprising the CMV sequence consisting of SEQ ID NO. 1 or a sequence having at least 85% identity thereto;

(b) A promoter region comprising a CMV sequence consisting of SEQ ID NO. 4 or a sequence having at least 85% identity thereto;

(c) A 5'UTR region, the 5' UTR region comprising, in 5 'to 3' order, a TPL sequence consisting of SEQ ID No. 11 or a sequence having at least 85% identity thereto and an eMLP sequence consisting of SEQ ID No. 12 or a sequence having at least 85% identity thereto;

(d) A coding sequence encoding the secreted polypeptide, wherein the coding sequence is operably linked to the promoter region;

(e) A second enhancer region comprising the entire EES sequence consisting of SEQ ID NO 13 or a sequence having at least 85% identity thereto; and

(f) 14 or a sequence having at least 85% identity thereto, and

optionally wherein the cassette does not comprise an RNA export signal.

12. The polynucleotide cassette of claim 11, further comprising each of SEQ ID NOs 76-80.

13. The polynucleotide cassette of claim 1, comprising 5 'to 3':

(a) A first enhancer region comprising the CMV sequence consisting of SEQ ID NO. 1 or a sequence having at least 85% identity thereto;

(b) A promoter region comprising the EF 1a sequence consisting of SEQ ID NO 3 or a sequence having at least 85% identity thereto;

(c) An intron region comprising the EF 1a sequence consisting of SEQ ID NO 5 or a sequence having at least 85% identity thereto;

(d) A 5' UTR region comprising a UTR2 sequence consisting of SEQ ID NO 6 or a sequence having at least 85% identity thereto;

(e) A coding sequence encoding the secreted polypeptide, wherein the coding sequence is operably linked to the promoter region;

(f) A second enhancer region comprising the 511-810EES sequence consisting of SEQ ID NO 7 or a sequence having at least 85% identity thereto;

(g) a WPRE RNA export sequence consisting of SEQ ID NO 8 or a sequence having at least 85% identity thereto; and

(h) A BGH polyadenylation site consisting of SEQ ID No. 9 or a sequence at least 85% identical thereto.

14. The polynucleotide cassette of claim 13, further comprising each of SEQ ID NOs 70-75.

15. The polynucleotide cassette of claim 1, comprising 5 'to 3':

(a) A first enhancer region comprising the CMV sequence consisting of SEQ ID NO. 1 or a sequence having at least 85% identity thereto;

(b) A promoter region comprising a CMV sequence consisting of SEQ ID NO. 4 or a sequence having at least 85% identity thereto;

(c) A 5'UTR region, the 5' UTR region comprising, in 5 'to 3' order, a TPL sequence and an eMLP sequence consisting of SEQ ID NO 11 and SEQ ID NO 12, respectively, or a sequence having at least 85% identity thereto;

(e) a coding sequence encoding a peptide or polypeptide;

(f) A second enhancer region comprising the 410-564EES sequence consisting of SEQ ID NO 16 or a sequence having at least 85% identity thereto;

(g) 17 or a sequence having at least 85% identity thereto; and

(h) A BGH polyadenylation site consisting of SEQ ID No. 9 or a sequence at least 85% identical thereto.

16. The polynucleotide cassette of claim 15, further comprising each of SEQ ID NOs 81-86.

17. The polynucleotide cassette of claim 1, comprising 5 'to 3':

(a) A first enhancer region comprising the CMV sequence consisting of SEQ ID NO. 1 or a sequence having at least 85% identity thereto;

(b) a promoter region comprising the actin sequence consisting of SEQ ID NO:96 or a sequence at least 85% identical thereto;

(c) A 5' UTR comprising an eMLP sequence consisting of SEQ ID No. 12 or a sequence having at least 85% identity thereto;

(e) A coding sequence encoding a peptide or polypeptide;

(f) A second enhancer region comprising the 511-810EES sequence consisting of SEQ ID NO 7 or a sequence having at least 85% identity thereto;

(g) 17 or a sequence having at least 85% identity thereto; and

(h) 20 or a sequence having at least 85% identity thereto.

18. the polynucleotide cassette of claim 17, further comprising each of SEQ ID NOs 87-91.

19. The polynucleotide cassette of claim 1, comprising 5 'to 3':

(a) A first enhancer region comprising the CMV sequence consisting of SEQ ID NO. 1 or a sequence having at least 85% identity thereto;

(b) A promoter region comprising a CMV sequence consisting of SEQ ID NO. 4 or a sequence having at least 85% identity thereto;

(c) An intron region comprising the CMVc sequence consisting of SEQ ID NO:18 or a sequence having at least 85% identity thereto;

(d) A 5' UTR region comprising a UTR1 sequence consisting of SEQ ID NO 19 or a sequence having at least 85% identity thereto;

(e) A coding sequence encoding a peptide or polypeptide;

(f) A second enhancer region comprising the entire EES sequence consisting of SEQ ID NO 13 or a sequence having at least 85% identity thereto;

(g) A WPRE RNA export sequence consisting of SEQ ID NO 8 or a sequence having at least 85% identity thereto; and

(h) 20 or a sequence having at least 85% identity thereto.

20. The polynucleotide cassette of claim 19, further comprising each of SEQ ID NOs 92-95.

21. A recombinant virus, comprising:

a) A capsid protein; and

b) A polynucleotide cassette according to any preceding claim.

22. The recombinant virus of claim 21, wherein the recombinant virus is a recombinant adeno-associated virus.

23. The recombinant virus of claim 22, wherein the capsid protein is an AAV variant 7m8 capsid protein or is derived from the AAV variant 7m8 capsid protein.

24. A pharmaceutical composition comprising the recombinant virus of any one of claims 21 to 23 and a pharmaceutically acceptable excipient.

25. An isolated host cell transfected or transduced with a polynucleotide cassette according to claim 1.

26. A method for expressing a transgene in a mammalian cell, the method comprising contacting one or more mammalian cells with an amount of the recombinant virus of any one of claims 21 to 23, wherein the secreted polypeptide is expressed in the one or more mammalian cells at a level, at least 5-fold higher than the level obtained by contacting said cell with a recombinant virus comprising a reference cassette encoding said secreted polypeptide, wherein the reference cassette comprises, in 5 'to 3' order, a CMV enhancer sequence (SEQ ID NO:2), a CMV promoter (SEQ ID NO:21), a chimeric intron (SEQ ID NO:22), a 5'UTR (SEQ ID NO:23), a coding sequence encoding the secreted polypeptide, a 3' UTR (SEQ ID NO:25), and an SV40polyA sequence (SEQ ID NO: 26).

27. A method for treating or preventing a disease in a mammal in need of disease treatment or prevention, the method comprising administering to the mammal an effective amount of a pharmaceutical composition according to claim 24.

28. The method of claim 27, wherein the disease is an ocular disease and the pharmaceutical composition is administered to the eye of the mammal.

29. The method of claim 28, wherein the pharmaceutical composition is administered to the eye of the mammal by intraocular injection or intravitreal injection.

30. The method of claim 29, wherein the ocular disease is selected from the group consisting of choroidal neovascularization and macular degeneration.

Technical Field

The present invention relates to gene therapy of disorders.

Background

One promising approach to the treatment and prevention of genetic diseases and other conditions is the delivery of therapeutic agents using gene delivery vehicles. Viral vectors are highly efficient gene transfer vehicles and can be used as gene delivery vehicles. In particular, adeno-associated virus (AAV) -based vectors are desirable vectors due to their non-integrating nature of the viral life cycle.

However, many challenges remain in designing polynucleotide cassettes and expression vectors for gene therapy. One significant challenge is to obtain adequate expression of the transgene in the target cell after gene transfer. In some cases, effective treatment of a disease or genetic disorder using a gene delivery vector (e.g., a recombinant virus) may depend on robust expression and efficient secretion of the therapeutic polypeptide. Thus, expression cassettes capable of driving high levels of secreted proteins from targeted (e.g., transduced) cells can be an important part of a therapeutically effective vector and successful gene therapy approach. Thus, there is a need for improved methods and optimized nucleic acid expression cassettes and vectors for expressing genes in mammalian cells.

The present invention meets this need.

disclosure of Invention

The present invention relates generally to the fields of molecular biology and virology, and in particular to gene expression cassettes and vectors comprising the same for delivering nucleic acid fragments encoding selected therapeutic constructs (e.g., peptides and polypeptides) to selected cells and tissues of vertebrates. These gene constructs are useful for the development of gene delivery vectors, including, for example, Herpes Simplex Virus (HSV), Adenovirus (AV), and AAV vectors for the treatment of diseases, disorders, and dysfunctions in mammals, and in particular in humans.

More particularly, the invention relates to gene expression cassettes for enhancing the expression of secreted proteins by eukaryotic or mammalian cells, and to methods of making and using such cassettes for use in both research and therapeutic applications, including but not limited to methods of treating diseases or disorders caused by or associated with defects, deficiencies, or loss of function of one or more proteins in a subject in need thereof.

The cassettes of the invention typically comprise a nucleic acid sequence or transgene encoding a polypeptide effective in treating a medical condition in a human or non-human animal subject. In some embodiments, the polypeptide is a secreted polypeptide. The cassette can be incorporated into a viral vector, such as an adeno-associated virus (AAV) vector, which can then be administered to eukaryotic or mammalian cells in vitro (e.g., in cell culture) or to a mammalian subject having or at risk of developing a medical condition.

The disclosed compositions can be used in a variety of research, diagnostic, and therapeutic protocols, including the prevention and treatment of human diseases. For example, the compositions of the invention may be used in the treatment of ocular diseases, angiogenesis-dependent diseases, diseases that respond to treatment with Vascular Endothelial Growth Factor (VEGF) inhibitors, or in enzyme replacement therapy, wherein the diseases are caused by or associated with defective or lost enzyme function.

Methods and compositions are provided for making polynucleotide expression cassettes and gene delivery vector compositions (e.g., viral vectors) including these expression cassettes for use in the preparation of medicaments useful for central and targeted gene therapy of diseases, disorders and dysfunctions in animals and, in particular, humans.

Embodiments of the invention include a non-naturally occurring polynucleotide cassette for enhancing transgene expression in a mammalian cell, the non-naturally occurring polynucleotide cassette comprising, in 5 'to 3' order: (a) a first enhancement sub-region; (b) a promoter region; (c) a coding sequence encoding a polypeptide gene product; (d) a second enhancement sub-region; and (e) a polyadenylation site. In some embodiments, the polynucleotide cassette further comprises a ribonucleic acid (RNA) export signal downstream of the second enhancer and upstream of the polyadenylation site. In one embodiment, the polynucleotide cassette is free of RNA export signals. In a more specific form, the cassette is free of an RNA export signal downstream of the second enhancer and upstream of the polyadenylation site.

In yet other embodiments, the polynucleotide cassette further comprises an intron downstream of the promoter and upstream of the coding sequence.

in still other embodiments, the polynucleotide cassette further comprises a5 'untranslated region (5' UTR) located upstream of the coding sequence and downstream of the promoter.

In a preferred embodiment, the polypeptide gene product is a secreted protein.

The polynucleotide cassette of the invention comprises a first enhancer region upstream of the promoter region. In a preferred embodiment, the first enhancer region comprises a Cytomegalovirus (CMV) sequence. In other embodiments, the first enhancer region comprises an elongation factor 1 α (EF1 α) sequence. In certain embodiments, the first enhancer region comprises a sequence having at least 85% identity to SEQ ID No. 1. In certain embodiments, the identity is at least 90%, at least 95%, or at least 99%.

The polynucleotide cassette of the invention comprises a promoter region comprising a promoter sequence or a functional fragment thereof. In some embodiments, the promoter region is specific for eukaryotic cells or more specifically for mammalian cells. In some embodiments, the promoter region comprises a promoter sequence selected from the group consisting of: the actin promoter, the Cytomegalovirus (CMV) promoter, the elongation factor 1 α (EF1 α) promoter, and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter. In preferred embodiments, the promoter region comprises a CMV promoter sequence or an EF 1a promoter sequence. In certain embodiments, the promoter region comprises a sequence having at least 85% identity to one of SEQ ID NOs 3, 4, or 96. In certain embodiments, the identity is at least 90%, at least 95%, or at least 99%.

In some embodiments, the polynucleotide cassette of the invention further comprises an intron region downstream of the promoter region. In some embodiments, the intron sequence is located downstream of the promoter and upstream of the 5' UTR. In some embodiments, the intron region comprises an elongation factor 1 α (EF1 α), actin, or CMVc sequence. In certain embodiments, the intron comprises a sequence having at least 85% identity to one of SEQ ID NOs 5, 18, or 97. In certain embodiments, the identity is at least 90%, at least 95%, or at least 99%.

The polynucleotide cassette of the invention comprises an untranslated region 5 'of the coding sequence, which is referred to herein as the 5' UTR. In some embodiments, the 5' UTR sequence is heterologous to the promoter sequence. The 5' UTR is located downstream of the promoter and upstream of the coding sequence. In some such embodiments, the 5' UTR comprises a sequence selected from the group consisting of: UTR1, UTR2, enhancer elements from the adenovirus major late promoter (eMLP), and tripartite leader sequence (TPL sequence) from adenovirus. In some embodiments, the 5' UTR comprises a UTR1 sequence. In one embodiment, the 5' UTR comprises UTR2 sequence. In a preferred embodiment, the 5' UTR comprises a TPL sequence and an eMLP sequence in 5' to 3' order. In some embodiments, the 5' UTR does not include a polynucleotide ATG. In certain embodiments, the 5' UTR comprises a sequence having at least 85% identity to one of SEQ ID NOs 6, 11, 12, or 19. In certain embodiments, the identity is at least 90%, at least 95%, or at least 99%.

Embodiments of the invention include polynucleotide cassettes for enhancing transgene expression. Accordingly, the polynucleotide cassette of the invention comprises coding sequences, which are also referred to as transgenes. In a particular embodiment, the polynucleotide cassette includes one coding sequence or one transgene and does not include two or more transgenes. The transgene may encode a therapeutic agent, such as, for example, a peptide or polypeptide. In a preferred embodiment, the transgene encodes a secreted polypeptide, which is also referred to herein as a secreted protein (secreted protein) or a secreted protein (secreted protein). Examples of secreted polypeptides that can be encoded by the coding sequence include, but are not limited to, soluble fms-like tyrosine kinase-1 (also known as sFLT-1), VEGF-binding fragments of sFLT-1, aflibercept (aflibercept), and alpha-1 antitrypsin or alpha 1-antitrypsin (A1 AT). In some embodiments, the secreted polypeptide is an anti-angiogenic polypeptide or an anti-vascular endothelial growth factor (anti-VEGF) polypeptide.

The subject expression cassettes provide for enhanced expression of the transgene product (e.g., polypeptide) in mammalian cells relative to expression of the transgene product in mammalian cells by the reference cassette. In preferred embodiments, the subject expression cassettes provide enhanced expression of the secreted protein in eukaryotic cells in vivo or in vitro (e.g., in cell culture or tissue explants) relative to expression of the secreted protein in eukaryotic cells in vivo or in vitro by the reference cassette. In some aspects, the eukaryotic cell is a mammalian cell. In a still further aspect, the mammalian cell is a human cell.

In some embodiments, expression of the secreted polypeptide by the polynucleotide cassette in the mammalian cell is at least about 2-fold, 3-fold, 5-fold, 9-fold, 10-fold, 20-fold, or 50-fold greater than expression of the secreted polypeptide by the reference cassette in the mammalian cell in vitro or in vivo.

In some embodiments, the expression level of the non-secreted polypeptide obtained by the polynucleotide cassette in a mammalian cell is about the same as, less than about 1.5 times the expression level, or less than about 2 times the expression level of the non-secreted polypeptide obtained by the reference cassette in a mammalian cell in vitro or in vivo. One non-limiting example of a non-secreted polypeptide is Green Fluorescent Protein (GFP).

According to some embodiments, the reference cassette referred to herein above and below (also referred to herein as a CMV reference control cassette) comprises, in 5 'to 3' order, a CMV enhancer sequence (SEQ ID NO:2), a CMV promoter (SEQ ID NO:21), a chimeric intron (SEQ ID NO:22), a 5'UTR (SEQ ID NO:23), a coding sequence encoding the peptide or polypeptide gene product, a 3' UTR (SEQ ID NO:25) and a SV40polyA sequence (SEQ ID NO: 26).

according to a specific embodiment, enhanced expression of the peptide or polypeptide is observed in an in vitro mammalian cell selected from the group consisting of a HeLa cell, a HEK-293 cell and an ARPE-19 cell. In another embodiment, the mammalian cell is contained in a retinal tissue explant.

In some forms, expression of a secreted polypeptide by a polynucleotide cassette in mammalian cells in vitro or in vivo (e.g., in a subject) is at least 2-fold, at least 5-fold, at least 10-fold, or about 5-fold to about 10-fold greater than expression of the secreted protein in the mammalian cells by the CMV reference control cassette under the same conditions, wherein the CMV reference control cassette comprises, in 5 'to 3' order, a CMV enhancer sequence shown in SEQ ID No. 2, a CMV promoter sequence shown in SEQ ID No. 21, a chimeric intron sequence shown in SEQ ID No. 22, a 5'UTR sequence shown in SEQ ID No. 23, a coding sequence encoding the secreted polypeptide, a 3' UTR sequence shown in SEQ ID No. 25, and an SV40polyA sequence shown in SEQ ID No. 26. In some embodiments, the polynucleotide cassette increases expression of the secreted protein in the mammalian cell by at least 2-fold (2X), 5-fold (5X), 10-fold (10X), or about 5-fold to about 10-fold as compared to expression of the protein in the mammalian cell by the CMV reference control cassette. In one embodiment, the polynucleotide cassette enhances or increases expression of a secreted protein in the transduced mammalian cell relative to expression of the protein in the transduced mammalian cell by the CMV reference control cassette.

The polynucleotide cassette includes a second enhancer region located downstream of the coding sequence and upstream of the polyadenylation signal sequence. When an RNA export signal sequence is present, the second enhancer sequence is located upstream of the RNA export signal (i.e., between the coding sequence and the RNA export signal), and the RNA export signal is located upstream of the polyadenylation signal. In a preferred embodiment, the second enhancer region comprises an EES selected from the group consisting of the complete Expression Enhancer Sequence (EES), 410-564EES and 511-810 EES.

In some embodiments, the polynucleotide cassette comprises an RNA export signal downstream of the second enhancer and upstream of the polyadenylation sequence (which is also referred to herein as a polyA sequence, polyA site or polyadenylation region). The RNA export signal may comprise a sequence selected from the group consisting of: human hepatitis b virus post-transcriptional element (HPRE) sequence and woodchuck hepatitis virus post-transcriptional element (WPRE) sequence. In certain embodiments, the RNA export signal comprises a sequence that is at least 85% identical to one of SEQ ID NOs 8 or 17. In certain embodiments, the identity is at least 90%, at least 95%, or at least 99%.

The polynucleotide cassette according to the invention comprises a polyadenylation site. The polyadenylation site may be located downstream of the second enhancer region or downstream of the RNA export region. In some embodiments, the polyadenylation region comprises a human growth hormone (HGH or HGH), bovine growth hormone (BGH or BGH), or a β -globin (β -globin) polyA sequence. In certain embodiments, the polyadenylation region comprises a sequence having at least 85% identity to one of SEQ ID NOs 9, 14 or 20 below. In certain embodiments, the identity is at least 90%, at least 95%, or at least 99%.

Some embodiments of the invention relate to a polynucleotide cassette for enhancing or increasing the expression of a secreted protein in a mammalian cell relative to the expression of the protein in the mammalian cell by a reference cassette, as measured by the amount (e.g., concentration or quantity) of the protein in the extracellular environment. The amount of protein in the sample can be measured by, for example, immunoassay.

In some embodiments, the polynucleotide cassette for enhancing transgene expression in a mammalian cell comprises, in 5 'to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 1); (b) a promoter region comprising the EF1 alpha sequence (SEQ ID NO: 3); (c) an intron region comprising the EF1 alpha sequence (SEQ ID NO: 5); (d) a 5' UTR region comprising a UTR2 sequence (SEQ ID NO: 6); (e) a coding sequence encoding a peptide or polypeptide; (f) a second enhancer region comprising the 511-810EES sequence (SEQ ID NO: 7); (g) WPRE RNA export sequence (SEQ ID NO: 8); and (h) a BGH polyadenylation site (SEQ ID NO: 9). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 70-75 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO 45 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO 46 or a sequence having at least 85% identity thereto.

In other embodiments, the polynucleotide cassette for enhancing transgene expression in a mammalian cell comprises, in 5 'to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 1); (b) a promoter region comprising a CMV sequence (SEQ ID NO: 4); (c) a 5' UTR region comprising, in 5' to 3' order, a TPL sequence and an eMLP sequence (SEQ ID NO:11 and SEQ ID NO:12, respectively); (d) a coding sequence encoding a peptide or polypeptide; (e) a second enhancer region comprising the entire EES sequence (SEQ ID NO: 13); and (f) an HGH polyadenylation site (SEQ ID NO: 14). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 76-80 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO 47 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID No. 48 or a sequence having at least 85% identity thereto.

In other embodiments, the polynucleotide cassette for enhancing transgene expression in a mammalian cell comprises, in 5 'to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 1); (b) a promoter region comprising a CMV sequence (SEQ ID NO: 4); (c) a 5' UTR region comprising, in 5' to 3' order, a TPL sequence and an eMLP sequence (SEQ ID NO:11 and SEQ ID NO:12, respectively); (e) a coding sequence encoding a peptide or polypeptide; (f) a second enhancer region comprising the 410-564EES sequence (SEQ ID NO: 16); (g) the HPRE RNA export sequence (SEQ ID NO: 17); and (h) a BGH polyadenylation site (SEQ ID NO: 9). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 81-86 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO 49 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO:50 or a sequence having at least 85% identity thereto.

in other embodiments, the polynucleotide cassette for enhancing transgene expression in a mammalian cell comprises, in 5 'to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 1); (b) a promoter region comprising the actin sequence (SEQ ID NO: 96); (c) a 5' UTR comprising an eMLP sequence (SEQ ID NO: 12); (e) a coding sequence encoding a peptide or polypeptide; (f) a second enhancer region comprising the 511-810EES sequence (SEQ ID NO: 7); (g) the HPRE RNA export sequence (SEQ ID NO: 17); and (h) a rabbit β -globin polyadenylation site (SEQ ID NO: 20). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 87-91 or sequences having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO 51 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO 52 or a sequence having at least 85% identity thereto.

In other embodiments, the polynucleotide cassette for enhancing transgene expression in a mammalian cell comprises, in 5 'to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 1); (b) a promoter region comprising a CMV sequence (SEQ ID NO: 4); (c) an intron region comprising the CMVc sequence (SEQ ID NO: 18); (d) a 5' UTR region comprising a UTR1 sequence (SEQ ID NO: 19); (e) a coding sequence encoding a peptide or polypeptide; (f) a second enhancer region comprising the entire EES sequence (SEQ ID NO: 13); (g) WPRE RNA export sequence (SEQ ID NO: 8); and (h) a rabbit β -globin polyadenylation site (SEQ ID NO: 20). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 92-95 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO:53 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID No. 54 or a sequence having at least 85% identity thereto.

In other embodiments, the polynucleotide cassette for enhancing transgene expression in a mammalian cell comprises, in 5 'to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 1); (b) a promoter region comprising the actin sequence (SEQ ID NO: 96); (c) an intron region comprising the chicken β -actin sequence (SEQ ID NO: 97); (d) a 5' UTR region comprising a UTR1 sequence (SEQ ID NO: 19); (e) a coding sequence encoding a peptide or polypeptide; (f) a second enhancer region comprising the 410-564EES sequence (SEQ ID NO: 16); (g) the HPRE RNA export sequence (SEQ ID NO: 17); and (h) a BGH polyadenylation site (SEQ ID NO: 9). In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 92-95 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 5' arm of the polynucleotide cassette comprises or consists of SEQ ID NO:39 or a sequence having at least 85% identity thereto. In certain of these embodiments, the 3' arm of the polynucleotide cassette comprises or consists of SEQ ID NO:40 or a sequence having at least 85% identity thereto.

In certain embodiments, the polynucleotide cassette comprises, or consists essentially of, in 5 'to 3' order: (a) a 5' arm; (b) a coding sequence encoding a peptide or polypeptide; and (c) a 3' arm. In certain of these embodiments, the 5' arm comprises a sequence selected from the group consisting of: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61 or a sequence having at least 85% identity thereto, and the 3' arm comprises a sequence selected from the group consisting of: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 62 or a sequence having at least 85% identity thereto. In particular embodiments, the 5 'arm and the 3' arm are SEQ ID NO 27 and 28, or SEQ ID NO 29 and 30, or SEQ ID NO 31 and 32, or SEQ ID NO 33 and 34, or SEQ ID NO 35 and 36, or SEQ ID NO 37 and 38, or SEQ ID NO 39 and 40, or SEQ ID NO 41 and 42, or SEQ ID NO 43 and 44, or SEQ ID NO 45 and 46, or SEQ ID NO 47 and 48, or SEQ ID NO 49 and 50, or SEQ ID NO 51 and 52, or SEQ ID NO 53 and 54, or SEQ ID NO 55 and 56, or SEQ ID NO 57 and 58, or SEQ ID NO 59 and 60, or SEQ ID NO 61 and 62, respectively.

In a preferred form of the invention, the peptide or polypeptide encoded by the coding sequence in the subject polynucleotide cassette is a peptide or polypeptide secreted or exported from the cell after it is expressed in the cell.

in some aspects of the invention, a gene delivery vector is provided that includes a polynucleotide cassette of the invention. In some embodiments, the gene delivery vector is a recombinant virus comprising: (a) a capsid protein; and (b) a polynucleotide cassette of the invention. In a more specific embodiment, the recombinant virus is a recombinant adeno-associated virus (AAV), wherein the recombinant adeno-associated virus comprises an AAV capsid protein and a polynucleotide cassette of the invention. In some embodiments, the AAV capsid protein is a wild-type AAV capsid protein. In other embodiments, the AAV capsid protein is a variant AAV capsid protein, wherein the variant capsid protein contains a substitution, deletion, or insertion of one or more amino acids relative to the parental capsid protein or a capsid protein derived therefrom. For example, in one embodiment, about 5 to about 11 amino acids are inserted into the insertion site of the GH loop or loop IV of the VP1 capsid protein relative to the corresponding parental AAV capsid protein. Suitable examples include an AAV variant capsid having a 7m8 variant capsid protein or a capsid protein derived from an AAV variant 7m8 capsid protein. In a particular example, the variant AAV comprises an aav2.7m8 capsid protein.

In some aspects of the invention, pharmaceutical compositions are provided that include a polynucleotide cassette of the invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a gene delivery vehicle of the invention and a pharmaceutical excipient.

One example is an isolated host cell transfected or transduced with a polynucleotide cassette of the present invention.

In some aspects of the invention, methods are provided for expressing a transgene in an in vitro or in vivo mammalian cell, the methods comprising contacting one or more in vivo or in vitro mammalian cells with an amount of a polynucleotide cassette, gene delivery vector or pharmaceutical composition of the invention, wherein the transgene is expressed in the one or more mammalian cells at a detectable level, or more preferably, at a level in the cell or cell culture that is about 2-fold, 5-fold or 10-fold (i.e., compared to the expression of the transgene by the reference CMV cassette) of the level obtained by a reference CMV cassette comprising, in 5 'to 3' order, an enhancer sequence (SEQ ID NO:2), a CMV promoter (SEQ ID NO:21), or a composition of the invention, measured under the same conditions (i.e., the other variables remain unchanged) Chimeric introns (SEQ ID NO:22), 5'UTR (SEQ ID NO:23), the transgene or coding sequence of interest, 3' UTR (SEQ ID NO:25), and SV40polyA sequence (SEQ ID NO: 26). Some embodiments provide a method for expressing a transgene in an in vitro or in vivo mammalian cell, the method comprising contacting one or more in vitro or in vivo mammalian cells with an amount of a recombinant virus of the invention, wherein the recombinant virus comprises a polynucleotide cassette according to the present disclosure. In preferred embodiments, the cassette or transgene encodes a secreted protein (also referred to herein as a secreted polypeptide) and expresses the secreted protein in the one or more mammalian cells at a level that is at least about 2-fold, 5-fold, 10-fold, 5-fold to 10-fold, or greater than 20-fold greater than the level obtained by contacting the cells with a recombinant virus comprising a CMV reference control cassette encoding the same protein. In some aspects, the mammalian cell is contacted with an amount of a polynucleotide cassette, pharmaceutical composition, or vector effective to alter one or more characteristics of the cell or effective to reduce one or more signs or symptoms of a disease in an individual.

In some aspects of the invention, methods are provided for treating or preventing a disease or disorder in a mammal in need of treatment or prevention of the disease or disorder. In some embodiments, the method comprises administering to the mammal an effective amount of a pharmaceutical composition of the invention, wherein the coding sequence encodes a therapeutic gene product. In one embodiment, the disease is an ocular disease and/or a disease associated with loss of function or a defect in a cellular protein product. In one embodiment, the ocular disease is associated with or caused by ocular neovascularization. One embodiment is a method for treating an ocular disease in a mammal in need thereof, the method comprising administering to an eye of a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition of the invention. In a more specific embodiment, a therapeutically effective amount of a pharmaceutical composition of the invention is administered to the eye of said individual in need of treatment by intraocular injection or by intravitreal injection.

ocular diseases for which the subject kits, compositions, and methods may be used include acute macular neuroretinopathy; behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular site or position; eye tumors; retinal disorders such as retinal vein occlusion, central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), Proliferative Vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitis retinal disease, and the like; sympathetic ophthalmia; vogt Koyanagi-Harada (VKH) syndrome; grape membrane diffusion; a posterior ocular condition caused by or affected by ocular laser therapy; a posterior ocular condition caused by or affected by photodynamic therapy; photocoagulation, radiation retinopathy; a condition of the epiretinal membrane; retinal vein branch obstruction; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinopathy of prematurity; retinitis pigmentosa; glaucoma, and glaucoma; uker (Usher) syndrome, cone rod dystrophy; sturgeon's (Stargardt) disease (fundus macular disease); hereditary macular degeneration; chorioretinal degeneration; leber's (Leber) congenital amaurosis; congenital stationary nyctalopia; choroideremia; Barde-Biedl syndrome; macular telangiectasia; leber's hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders including achromatopsia, achromatopsia and achromatopsia.

Drawings

A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Figure 1 shows a series of polynucleotide cassettes constructed to assess transgene expression. The regulatory elements and coding sequences (i.e., genes) present in each cassette are listed in 5 'to 3' order, left to right. The cartridge is identified by a cartridge number, for example, cartridge number 11 may be referred to as cartridge number 11, cartridge 11, or simply C11.

Figure 2 shows a graph comparing the expression of aflibercept (secreted protein) by the polynucleotide cassette after transfection into HeLa cells. The expression levels are plotted against the expression levels obtained from the "base plasmid" depicted in FIG. 1.

Figure 3 shows the expression of aflibercept (secreted protein) by the selected polynucleotide cassette after transduction into HEK293 cells.

Figures 4A and 4B show the expression of aflibercept (secreted protein) in transduced porcine retina explants by selected polynucleotide cassettes one week (figure 4A) and two weeks (figure 4B) after transduction. Each cassette was packaged in 7m8 capsid (variant of AAV 2). Background signal associated with the assay was determined with vehicle (buffer only) non-viral controls.

FIG. 5 depicts a comparison of sFLT-1 expression in transfected ARPE-19 cells from each polynucleotide cassette construct comprising the reference cassette (CMV-sFLT 1). The sequences encoding sFLT-1 in boxes 10, 11 and 12 were codon optimized ("CO"). The base plasmid had codon optimized sFLT1 only under the ubiquitous promoter, without any other regulatory elements.

FIG. 6 depicts a comparison of sFLT-1 expression in transfected HEK293 cells by the respective constructs.

FIG. 7 depicts a comparison of sFLT-1 expression in transfected HeLa cells by each construct (containing the reference cassette (CMV-sFLT 1)).

Figure 8 shows the expression of Green Fluorescent Protein (GFP) in transfected HeLa cells by polynucleotide cassettes C7, C11 and C13 compared to the expression of GFP by a CMV reference control cassette and to a vehicle (buffer only) control.

FIG. 9 depicts a comparison of sFLT-1 expression by individual polynucleotide cassettes in transduced HEK293 cells. Data represent the amount of sFLT-1 in the cell culture supernatant three days after transduction with 7m8 vector. In each case, the carrier packages the designated cassettes. Expression of sFLT-1 by cassettes C10 and C11 (as measured in cell culture supernatant) was compared to expression of sFLT-1 by the CMV reference cassette. Background signal was determined with vehicle (buffer only) control. As an additional control, cells were transduced with recombinant AAV2 vector containing a CMV-sFLT1 reference control cassette. In the first experiment, the amount of sFLT1 secreted from cassette 11 was higher than the immunoassay range, so further dilution and analysis had to be performed.

Fig. 10A and 10B show the expression of sFLT1 by selected polynucleotide cassettes in transduced porcine retina explants. sFLT1 expression was measured in the supernatant (fig. 10A) as well as in the tissue lysates (fig. 10B).

FIG. 11 shows an illustrative example of a recombinant plasmid containing cassette 11. The cassette is flanked by Inverted Terminal Repeats (ITRs) from adeno-associated virus serotype 2(AAV 2).

Detailed Description

the present disclosure provides polynucleotide cassettes and expression vectors for expressing genes in cells. Also provided are pharmaceutical compositions and methods of using any of the compositions to promote expression of a gene in a cell (e.g., in an individual), e.g., to treat or prevent a disorder. These and other objects, advantages and features of the invention will become apparent to those skilled in the art upon a reading of the details of the compositions and methods as more fully described hereinafter.

Definition of

A "non-naturally occurring" polynucleotide cassette is one that is not found in nature.

A "secreted protein" or "secreted polypeptide," also referred to herein as a "secreted protein," is any protein secreted by or exported from a living cell. One non-limiting example of a secreted protein for use with the presently described polynucleotide cassette is sFLT-1.

The terms "disease," "disorder," and "medical condition" are synonymous and are used interchangeably herein.

As used herein, "carrier" refers to a macromolecule or association of macromolecules that includes or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors (i.e., viruses such as adeno-associated virus), liposomes, and other gene delivery vehicles.

the term "AAV" is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or derivatives thereof. The term encompasses all subtypes as well as naturally occurring and recombinant forms, unless otherwise required. The term "AAV" encompasses AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "primate AAV" refers to AAV infecting a primate, "non-primate AAV" refers to AAV infecting a non-primate mammal, and "bovine AAV" refers to AAV infecting a bovine mammal, and the like.

An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically composed of all capsid proteins of a wild-type AAV) and an encapsidation polynucleotide. If the particle includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is often referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotide is flanked by AAV Inverted Terminal Repeats (ITRs).

As used herein, the term "replication defective" with respect to an AAV viral vector of the present invention means that the AAV vector is unable to independently replicate and package its genome. For example, when a subject's cells are infected with rAAV virions, the heterologous gene is expressed in the infected cells, however, rAAV cannot replicate further due to the fact that the infected cells lack AAV rep and cap genes and helper function genes.

As used herein, an "AAV variant" or "AAV mutant" refers to a viral particle comprised of a variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a corresponding parental AAV capsid protein, and wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of retinal cells of an AAV virion comprising the corresponding parental AAV capsid protein, wherein the AAV capsid protein does not comprise the amino acid sequences present in naturally occurring AAV capsid proteins. The polynucleotide expression cassettes of the present disclosure can be packaged in variant AAV particles to facilitate delivery of the cassette to a particular cell type (e.g., retinal cells) in a target tissue.

As used herein, the term "gene" or "coding sequence" refers to a nucleotide sequence that encodes a gene product in vitro or in vivo. The term "transgene" refers to a coding sequence or gene that is delivered into a cell by a vector. The coding sequence or gene may encode a peptide or polypeptide molecule.

As used herein, "therapeutic gene" and "therapeutic protein" refer to a gene or protein that, when expressed, confers a beneficial effect on the cell or tissue or mammal in which it is present, in which it is expressed. Examples of beneficial effects can be alleviation or amelioration of signs or symptoms of a condition or disease, prevention or inhibition of a condition or disease, or imparting a desired characteristic. Therapeutic genes and proteins include genes and proteins that correct genetic defects in a cell or mammal.

A "therapeutically effective amount" or "effective amount" of a polynucleotide cassette, recombinant virus, or pharmaceutical composition of the invention is an amount sufficient to cause a reduction in one or more signs or symptoms of a disease or medical condition in a subject, wherein the subject can be a human or non-human mammal.

As used herein, the term "gene product" refers to the desired expression product of a polynucleotide sequence (e.g., a peptide or protein).

As used herein, the terms "polypeptide" and "protein" refer to polymers of amino acids of any length. The term "peptide" refers to a polymer of amino acids of about 50 amino acids or less. The term also encompasses amino acid polymers that have been modified as by, for example, disulfide bond formation, glycosylation, lipidation, or phosphorylation. In some examples, the subject polypeptides may be greater than 50 amino acids in length.

"comprising" means that the listed elements are, for example, essential in the compositions, methods, kits, etc., but that other elements may be included to form, for example, the compositions, methods, kits, etc., within the scope of the claims. For example, an expression cassette that "comprises" a gene encoding a therapeutic polypeptide operably linked to a promoter is one that may contain elements other than the gene and promoter (e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.).

"consisting essentially of … …" is intended to mean a limitation on the scope of a particular material or step described, e.g., as a composition, method, kit, etc., that does not materially affect one or more of the basic and novel characteristics of the composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may comprise additional sequences, e.g., linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" the recited sequence has an amino acid sequence of the sequence plus or minus about 10 amino acid residues at the boundaries of the sequence based on the full-length untreated polypeptide from which it is derived, e.g., 10, 9, 8, 7,6, 5,4, 3,2, or 1 fewer than the binding amino acid residues or 1, 2,3, 4,5, 6, 7,8, 9, or 10 more residues than the binding amino acid residues.

"consisting of … …" means that any element, step, or ingredient not specified in the claims is excluded from the composition, method, or kit. For example, an expression cassette "consisting of" a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence consists of only the promoter, the polynucleotide sequence encoding the therapeutic polypeptide, and the polyadenylation sequence. As another example, a polypeptide "consisting of" a recited sequence contains only the recited sequence.

As used herein, "expression vector" encompasses vectors, e.g., plasmids, minicircles, viral vectors, liposomes, etc., that comprise the subject polynucleotide cassettes encoding the gene products of interest, and are used to deliver the subject polynucleotides to the intended target cells.

As used herein, "promoter" encompasses a DNA sequence that directs RNA polymerase to bind and thereby promote RNA synthesis. Promoters and corresponding protein or polypeptide expression may be ubiquitous (meaning having strong activity in a wide range of cells, tissues and species) or cell type specific, tissue specific or species specific. Promoters may be "constitutive" (meaning persistently active) or "inducible" (meaning that the promoter may be activated or inactivated by the presence or absence of a biological or non-biological agent). Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences, which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5 'region or the 3' region of the native gene.

As used herein, "enhancer" encompasses cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also referred to as "silencers". Enhancers can function in either orientation at a distance of a few thousand base pairs (kb) from the coding sequence and a position downstream of the transcribed region (i.e., can be related to the coding sequence).

As used herein, "polyadenylation signal sequence" encompasses the recognition region required for endonuclease cleavage of an RNA transcript, followed by the polyadenylation consensus sequence AATAAA. The polyadenylation signal sequence provides a "polyA site", i.e., a site on the RNA transcript to which adenine residues will be added by post-transcriptional polyadenylation.

As used herein, the term "operably linked" refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, termination signal sequences, polyadenylation sequences, etc.) in a relationship permitting them to operate in the intended manner. For example, a promoter is operably linked to a coding region if it helps to initiate transcription of the coding sequence. Intervening residues may be present between the promoter and the coding region so long as this functional relationship is maintained.

As used herein, the term "heterologous" refers to an entity that is of a different genotype from the rest of the entity to which it is compared. For example, polynucleotides introduced into plasmids or vectors derived from different species by genetic engineering techniques are heterologous polynucleotides. As another example, a promoter removed from its native coding sequence and operably linked to a coding sequence not found in nature is a heterologous promoter. Thus, for example, a rAAV comprising a heterologous nucleic acid encoding a heterologous gene product is a rAAV comprising nucleic acid not normally comprised in a naturally-occurring wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring wild-type AAV.

As used herein, the term "endogenous" with respect to a nucleotide molecule or gene product refers to a nucleic acid sequence (e.g., a gene or genetic element) or gene product (e.g., RNA, protein) that naturally occurs in or is associated with a host virus or cell.

As used herein, the term "native" refers to a nucleotide sequence (e.g., a gene) or a gene product (e.g., RNA, protein) that is present in a wild-type virus or cell.

As used herein, the term "variant" refers to a mutant of a reference polynucleotide or polypeptide sequence, e.g., a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity to the reference polynucleotide or polypeptide sequence. In other words, a polypeptide variant includes at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polypeptide sequence (e.g., a native polypeptide sequence), and a polynucleotide variant includes at least one nucleotide or nucleoside difference (e.g., nucleotide or nucleoside substitution, insertion, or deletion) relative to a reference polynucleotide sequence (e.g., a native polynucleotide sequence).

As used herein, the term "sequence identity" or "percent identity" refers to the degree of identity between two or more polynucleotides when aligned using a nucleotide sequence alignment program; or the degree of identity between two or more polypeptide sequences when aligned using an amino acid sequence alignment program. Similarly, the term "identical" or percent "identity," when used in the context of two or more nucleotides or amino acid sequences, refers to two sequences that are the same or have a particular percentage of amino acid residues or nucleotides when compared and aligned for maximum correspondence, e.g., as measured using a sequence comparison algorithm (e.g., Smith-Waterman algorithm, etc.) or by visual inspection. For example, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970, journal of molecular biology (J.mol. biol.), 48:444-453) algorithm, which has been incorporated into the GAP program in the GCG software package, using either the Blossum62 matrix or the PAM250 matrix with GAP weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2,3, 4,5, or 6. As another example, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the nwsgapdna. cmp matrix and GAP weights of 40, 50, 60, 70, or 80 and length weights of 1, 2,3, 4,5, or 6. A particularly preferred set of parameters (which should be used unless otherwise specified) is the Blossum62 scoring matrix, a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of e.meyers and w.miller (1989, computer applications in bioscience (cabaos), 4:11-17), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as "query sequences" to search public databases to, for example, identify other family members or related sequences. This search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al (1990, journal of molecular biology, 215: 403-10). A BLAST nucleotide search can be performed using NBLAST program (score 100, word length 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program (score 50, word length 3) to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al (1997, Nucleic Acids Res., 25: 3389-. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

As used herein, the terms "biological activity" and "biological activity" refer to an activity attributed to a particular biological element in a cell. For example, "biological activity" of an "immunoglobulin", "antibody" or fragment or variant thereof refers to the ability to bind an antigenic determinant and thereby promote immune function. As another example, the biological activity of a polypeptide or functional fragment or variant thereof refers to the ability of the polypeptide or functional fragment or variant thereof to perform its native function, e.g., binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element (e.g., promoter, enhancer, kozak sequence, etc.) refers to the ability of the regulatory element, or a functional fragment or variant thereof, respectively, to regulate the expression of (i.e., promote, enhance, or activate translation of) a gene to which it is operably linked.

As used herein, the term "administration" or "introducing" refers to the delivery of a vector for recombinant protein expression to a cell, a cell and/or an organ of a subject, or a subject. Such administration or introduction may occur in vivo, in vitro, or ex vivo. The vector for expressing the gene product can be introduced into the cell by transfection, which generally means insertion of heterologous DNA into the cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection); or by infection or transduction, which generally refers to the introduction of a nucleic acid molecule into a cell by an infectious agent (i.e., a virus or viral vector).

In general, a cell is referred to as "transduced", "infected", "transfected" or "transformed" according to the method used to administer, introduce or insert the heterologous DNA (i.e., vector) to the cell. When DNA is introduced into a cell by a virus or viral vector, the cell is transduced with exogenous or heterologous DNA. When DNA is introduced into a cell by non-viral methods, the cell is transfected with exogenous or heterologous DNA. Non-viral methods include chemical methods (e.g., lipofection) and non-chemical methods. The terms "transduced" and "infected" are used interchangeably herein to refer to a cell that has received heterologous DNA or a heterologous polynucleotide from a virus or viral vector.

As used herein, the term "host cell" refers to a cell that has been transduced, infected, transfected or transformed with a vector. The vector may be a plasmid, a viral particle, a phage, or the like. Culture conditions (e.g., temperature, pH, etc.) are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. It is understood that the term "host cell" refers to the originally transduced, infected, transfected or transformed cell and its progeny.

The terms "treatment" and "treating" refer to the alleviation of one or more signs or symptoms of a disease or disorder.

"ocular disease" refers to a disease, illness, or condition that affects or involves the eye or one or more portions or regions of the eye. Thus, ocular diseases include retinal diseases or diseases that affect the light sensitive layer of the posterior tissues of the eye. The eye contains the eyeball and the tissues and fluids that make up the eyeball, the periocular muscles (e.g., the oblique and rectus muscles), and the portion of the optic nerve within or near the eyeball.

A tissue "explant" is a piece of tissue that has been transferred from an animal to a nutrient medium.

The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to a mammal, including but not limited to: human and non-human primates, including apes and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

Various compositions and methods of the invention are described below. Although specific compositions and methods are exemplified herein, it should be understood that any of a number of alternative compositions and methods are suitable and suitable for practicing the present invention. It will also be appreciated that the expression constructs and methods of the invention can be evaluated using standard procedures in the art.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well described in the literature, e.g., molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (edited by m.j. gate, 1984); animal Cell Culture (Animal Cell Culture), edited by r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Academic Press, Inc.; handbook of experimental immunology (edited by d.m.weir and c.c.blackwell); gene Transfer Vectors for Mammalian Cells (Gene Transfer Vectors for Mammarian Cells) (edited by J.M.Miller and M.P.Calos, 1987); current Protocols in Molecular Biology (edited by F.M. Ausubel et al, 1987); PCR: polymerase Chain Reaction (PCR: The Polymerase Chain Reaction), ed (edited by Mullis et al, 1994); and Current Protocols in Immunology (J.E. Coligan et al, 1991), each of which is expressly incorporated herein by reference.

several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One skilled in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "includes", "including", "includes", "having", "has", "with", or variants thereof are used in the detailed description and/or the claims, such terms are intended to be inclusive (in a manner similar to the term "comprising").

The terms "about" or "approximately" mean that the particular value determined by one of ordinary skill in the art is within an acceptable error range, which will depend in part on how the value is measured or determined, i.e., limited by the measurement system. For example, "about" can mean within 1 or a standard deviation of greater than 1, according to practice in the art. Alternatively, "about" or "approximately" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and more preferably still up to 1% of a given value. Alternatively, particularly for biological systems or methods, the term may mean within an order of magnitude of a value, preferably within 5-fold and more preferably within 2-fold of the value. When particular values are described in the present application and claims, unless otherwise specified, it should be assumed that the term "about" means within an acceptable error range for the particular value.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be understood that in case of conflict, the present disclosure supersedes any disclosure incorporated herein.

It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," etc. in connection with the recitation of claim elements, or use of a "negative type" limitation.

The disclosure in the publications discussed herein is provided solely for their purpose of illustration prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. In addition, the dates of publication provided may be different from the actual publication dates which may need to be independently determined.

Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art and the practice of the present invention will employ conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of one skilled in the art.

Composition comprising a metal oxide and a metal oxide

in some aspects of the disclosure, compositions for expressing a transgene in one or more eukaryotic cells are provided. In some aspects, the eukaryotic cell is a mammalian cell. In some aspects, the mammalian cell is a retinal cell, such as a retinal ganglion cell, amacrine cell, horizontal cell, bipolar cell, photoreceptor cell, cone cell, rod cell, muller glial cell, or retinal pigment epithelial cell.

In some embodiments of the disclosure, the composition is a polynucleotide cassette. "polynucleotide cassette" refers to a polynucleotide sequence, e.g., regulatory elements, translation initiation sequences, coding sequences, termination sequences, etc., typically operably linked to each other that includes two or more functional polynucleotide sequences. Typically, the subject polynucleotide sequences are comprised of DNA. Likewise, "polynucleotide cassette for expressing a transgene in a mammalian cell" refers to a combination of two or more functional polynucleotide sequences (e.g., promoters, enhancers, 5' UTRs, switch initiation sequences, coding sequences, termination sequences, etc.) that facilitate expression of the transgene in the cell.

For example, in some embodiments, the polynucleotide cassette comprises, in 5 'to 3' order: (a) optionally, a first enhancer region; (b) a promoter region, wherein the promoter region is specific for a eukaryotic cell; (c) a coding sequence encoding a polypeptide gene product; (d) a second enhancement sub-region; and (e) a polyadenylation site. In still other embodiments, the polynucleotide cassette further comprises a5 'untranslated region (5' UTR) upstream of the coding sequence. In still other embodiments, the polynucleotide cassette further comprises an intron region downstream of the promoter and upstream of the coding sequence. In other embodiments, the polynucleotide cassette further comprises an RNA export signal downstream of the second enhancer and upstream of the polyadenylation site. With respect to the polynucleotide cassettes disclosed herein, it is understood that the coding sequence is operably linked to expression control sequences in the cassette. For example, a coding sequence is operably linked to a promoter region, one or more enhancer regions, and a polyadenylation site.

In some embodiments, the polynucleotide cassettes of the present disclosure provide for enhanced expression of a transgene in a mammalian cell. In certain embodiments, the arrangement of two or more functional polynucleotide sequences within a polynucleotide cassette of the present disclosure provides for enhanced expression of a transgene in a mammalian cell. By "enhanced" is meant that expression of the transgene is increased, enhanced, or greater in a cell carrying a polynucleotide cassette of the present disclosure relative to a cell carrying a transgene operably linked to a comparable regulatory element. In other words, transgene expression by a polynucleotide cassette of the present disclosure is increased, enhanced, or stronger relative to expression by a polynucleotide cassette that does not include one or more optimized elements of the present disclosure (i.e., a reference control cassette, such as a CMV reference control cassette described herein). In certain embodiments, the expression enhancement is specific for or limited to one or more desired cell types. In one embodiment, the transgene encodes a protein that is secreted by the cell into the aqueous environment surrounding the cell.

for example, expression of a transgene in a cell comprising a polynucleotide cassette comprising a promoter disclosed herein can be enhanced, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different promoter. As another example, expression of a transgene in a cell comprising a polynucleotide cassette comprising an enhancer sequence disclosed herein may be enhanced, or increased, enhanced, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different enhancer sequence. As another example, expression of a transgene in a cell comprising a polynucleotide cassette encoding a 5'UTR disclosed herein can be enhanced, or increased, potentiated, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different 5' UTR coding sequence. As another example, expression of a transgene in a cell comprising a polynucleotide cassette comprising an intron disclosed herein can be enhanced, or increased, potentiated, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different intron sequence. In yet another example, expression of the transgene in a cell comprising a polynucleotide cassette comprising an intron disclosed herein can be enhanced, or increased, potentiated, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a reference control cassette (such as a CMV reference control cassette disclosed herein).

In preferred embodiments, the polynucleotide expression cassette facilitates expression of the transgene (or higher level of expression compared to a reference cassette) in one or more specific cell or tissue types in vitro and in vivo. Examples of cell types include, but are not limited to, HeLa cells, HEK-293 cells, ARPE-19 cells (human retinal pigment epithelial cell line), retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Muller glia cells, and retinal pigment epithelial cells. In another embodiment, increased expression is observed in cells of a retinal tissue explant.

In some embodiments, expression of the secreted polypeptide by the polynucleotide cassette in the mammalian cell is at least about 2-fold, 3-fold, 5-fold, 9-fold, 10-fold, 20-fold, or 50-fold greater than expression of the secreted polypeptide by the reference cassette in the mammalian cell in vitro or in vivo. More typically, the expression of the secreted polypeptide is 2 to 10 fold, 5 to 10 fold, 9 to 10 fold, at least 2 fold, at least 5 fold, or at least 10 fold greater than the expression of the polypeptide in the mammalian cell by the reference cassette. In other words, the polynucleotide cassette expresses the secreted protein in mammalian cell culture at a level that is at least or greater than 2-fold, 5-fold, 10-fold, 50-fold, or about 5-fold to about 10-fold greater than the level of expression obtained by the reference cassette in mammalian cell culture.

Without wishing to be bound by theory, it is believed that enhanced expression of the transgene in the intracellular or extracellular environment (e.g., culture supernatant or tissue matrix) results from faster accumulation of the gene product in the cell or a more stable gene product in the cell. Thus, enhanced transgene expression by the polynucleotide cassettes of the present disclosure can be observed in a variety of ways. For example, if the transgene is operably linked to comparable regulatory elements (such as those in the CMV reference control cassettes described herein), enhanced expression can be observed by detecting expression of the transgene, followed by detecting exposure of the polynucleotide cassette to the cell earlier than expressed (e.g., 7 days, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks or more earlier than expressed). Enhanced expression with increasing amounts of gene product per cell can also be observed. For example, the amount of gene product per mammalian cell may be increased 2-fold or more, e.g., increased 3-fold or more, increased 4-fold or more, increased 5-fold or more, or increased 10-fold or more. Enhanced expression may also be observed with increasing numbers of mammalian cells expressing detectable levels of the transgene carried by the polynucleotide cassette. For example, the number of mammalian cells expressing detectable levels of the transgene may be increased 2-fold or more, e.g., increased 3-fold or more, increased 4-fold or more, increased 5-fold or more, or increased 10-fold or more. As another example, polynucleotides of the invention can promote detectable levels of transgenes in a greater percentage of cells as compared to conventional polynucleotide cassettes; for example, while a conventional cassette may promote a detectable level of transgene expression in, e.g., less than 5% of the cells in a region, a polynucleotide of the invention promotes a detectable level of expression in 5% or more of the cells in the region; for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, in some cases 50% or more, 55% or more of the contact; 60% or more, 65% or more, 70% or more, 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more) of the cells will express a detectable level of the gene product. Enhanced expression with altered cell viability and/or function may also be observed.

The polynucleotide cassettes of the present disclosure typically include a promoter region. In certain embodiments, the promoter region promotes expression of the coding sequence in mammalian cells. In some cases, the promoter is a ubiquitous promoter, i.e., it is a promoter that is active in a wide range of cells, tissues and species. Suitable examples include actin, Cytomegalovirus (CMV), elongation factor 1 α (EF1 α), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoters.

in some embodiments, the polynucleotide comprises one or more enhancers. Enhancers are nucleic acid elements that enhance transcription. In some embodiments, the polynucleotide cassette comprises a first enhancer upstream of the coding sequence and a second enhancer downstream of the coding sequence. Suitable exemplary enhancers include, but are not limited to, EF1 α, CMV, intact EES or portions thereof, such as 410-564EES or 511-810 EES. EES (expression enhancer sequence) corresponds to the human scaffold attachment region of human interferon-beta or SAR (Agarwal, M et al (1998) "enhancement of retroviral vector expression in primary T cells mediated by the scaffold attachment region" [ J. virol.) ] 72 (5: 3720-. In certain embodiments, the upstream enhancer includes, but is not limited to, EF 1a or CMV. In certain embodiments, the downstream enhancer includes, but is not limited to, the complete expression enhancer sequence (complete EES), 410-564EES or 511-810 EES.

In some embodiments, the subject polynucleotide cassettes include sequences encoding the 5' untranslated region, i.e., polynucleotide sequences encoding the 5' untranslated region of a coding sequence, also referred to as the 5' UTR. In some embodiments, the 5' UTR does not contain a polynucleotide ATG. Suitable exemplary 5' UTR sequences include, but are not limited to, sequences selected from: i) triple leader sequence (TPL) from Adenovirus (Logan, J et al (6 1984) "triple leader sequence of Adenovirus enhances translation of mRNA in the late stage of infection (Adenoviral leader sequences of mRNAs later infection)", (Proc. Natl. Acad. Sci. USA) 81: 3655-; ii) enhancer element sequences from the adenovirus major late promoter (eMLP) (Durocher, Y et al (2002) "High-level and High-throughput recombinant proteins produced by transient transfection of human 293-EBNA1cells grown in suspension by High-level and High-throughput recombinant protein production by transformation of sub-growth human 293-EBNA1 cells", "nucleic acid research (nucleic acid. Res. 30(2): e 9); (iii) UTR 1; and (iv) UTR 2. In a preferred embodiment, the 5' UTR comprises in 5' to 3' order TPL and eMLP sequences.

In some embodiments, the subject polynucleotide cassettes further comprise an intron comprising a splice donor/acceptor region. In some embodiments, the intron is located downstream of the promoter region and upstream of the translation initiation sequence of the gene. Introns are DNA polynucleotides that are transcribed into RNA by intron splicing and are removed during mRNA processing. Expression of polynucleotide cassettes containing introns is generally higher than expression of those polynucleotide cassettes without introns. Introns can stimulate expression between 2-fold and 500-fold (Buchman and Berg,1988, Mol Cell Bio, 8(10): 4395). The efficiently spliced intron contains a pre-splice donor, a branch point and a Py-rich region (Senapathy et al, 1990; "methods in enzymology" (meth. enzymol.) 183,252-78; Wu and Krainer, 1999; "molecular cell biology", 19(5): 3225-36). The 5 'intron is generally more effective than the intron at the 3' end (Huang and Gorman, 1990; "molecular cell biology", 10: 1805). While introns are known to generally increase gene expression levels, the specific increase (if any) of a given cDNA is empirical and must be tested; for example, chimeric introns in the pSI vector increased CAT expression 21-fold, but luciferase expression only 3-fold. Exemplary intron sequences include, but are not limited to, sequences from actin, elongation factor 1 α (EF1 α), enhancer element from adenovirus major late promoter (eMLP), and CMVc.

The coding sequence to be expressed in a cell may be any polynucleotide sequence, for example, a gene or cDNA encoding a gene product, such as a polypeptide. The coding sequence may be heterologous to the promoter sequence and/or 5'UTR sequence to which said coding sequence is operably linked, i.e. said coding sequence is not naturally operably associated with said promoter or 5' UTR. Alternatively, the coding sequence may be endogenous to a promoter sequence and/or 5'UTR sequence to which the coding sequence is operably linked, i.e. the coding sequence is naturally associated with the promoter or 5' UTR. The gene product may act internally on the mammalian cell, or it may act externally, e.g., it may be secreted. For example, when the transgene is a therapeutic gene, the coding sequence can be any gene that encodes a desired gene product, or a functional fragment or variant thereof, which can be used as a therapeutic agent for treating a disease or disorder. Thus, the coding sequence in the polynucleotide cassette can encode, for example, an opsin protein or a protein that inhibits VEGF, or the polynucleotide can encode a protein or enzyme effective to reduce one or more signs or symptoms of a disease.

in various preferred embodiments, the transgene encodes a peptide or protein that is secreted from the cell. In some embodiments, the secreted protein is a therapeutic protein or a protein effective to treat a disease in a subject. In some embodiments, the therapeutic protein is an anti-angiogenic polypeptide or a polypeptide that inhibits the growth of new blood vessels (angiogenesis). In some forms, the secreted protein is an anti-VEGF protein or a protein that inhibits Vascular Endothelial Growth Factor (VEGF). Examples of anti-VEGF proteins include ranibizumab (ranibizumab), bevacizumab (bevacizumab), and aflibercept. Another example of an anti-VEGF polypeptide is soluble fms-like tyrosine kinase-1 (sFLT-1). In other cases, the secreted protein includes or consists of a VEGF-binding protein or a functional fragment thereof (as any one disclosed in U.S. patent nos. 5,712,380, 5,861,484, and 7,071,159), or a VEGF-binding fusion protein as disclosed, for example, in U.S. patent No. 7,635,474. In some forms, the secreted protein comprises or consists of a single chain antibody (such as, for example, a single chain anti-VEGF antibody). According to one embodiment, the transgene encodes sFLT-1, and in a more specific embodiment, the transgene encodes human sFLT-1. Alternatively, the transgene may comprise a sequence encoding a functional VEGF-binding fragment of sFLT-1 (Wiesmann et al, 1997; Cell (Cell), 91: 695-. According to another example, the transgene encodes A1AT or alpha-1 antitrypsin (Chiuchiolo et al, 2013,24(4): 161-.

sFLT-1 is a soluble truncated form of the VEGF receptor FLT-1 and is also known as soluble vascular endothelial growth factor receptor-1 (sVEGFR-1). Recombinant sFLT-1 binds and inhibits VEGF (Kendall and Thomas, 1993; Proc. Natl. Acad. Sci. USA 90(22): 10705-10709). In nature, recombinant sFLT-1 is produced by alternative mRNA splicing and lacks the membrane proximal immunoglobulin-like domain, transmembrane region (transmembrane spanning region), and intracellular tyrosine kinase domain. As described herein, "soluble" FLT-1 or sFLT-1 refers to FLT-1, said FLT-1 is not limited to a cell membrane. Unbound sFLT-1 can diffuse freely in the extracellular space or in solution.

In one embodiment of the invention, the transgene coding sequence is modified or "codon optimized" to enhance expression by replacing infrequently represented codons with more frequently represented codons. The coding sequence is a portion of the mRNA sequence that encodes the amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species will utilize trnas that encode the same amino acid at different frequencies (each carrying an anticodon). When a gene sequence contains codons that are infrequently represented by the corresponding tRNA, the ribosomal translation mechanism may slow, thereby preventing efficient translation. Expression may be improved by "codon optimisation" of a particular species in which the coding sequence is altered to encode the same protein sequence using codons highly expressed and/or used by highly expressed human proteins (Cid-Arregui et al, 2003; "journal of virology" 77: 4928). In one aspect, the coding sequence is optimized for translation in primates. In one aspect of the invention, the coding sequence of the transgene is modified to replace codons that are not frequently expressed in mammals or primates with codons that are frequently expressed in primates. For example, in some embodiments, the coding sequence encoded by the transgene encodes a polypeptide having at least 85% sequence identity, e.g., at least 90% sequence identity, e.g., at least 95% sequence identity, at least 98% identity, at least 99% identity, to a polypeptide encoded by a sequence disclosed above or herein, wherein at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon in the sequence disclosed above or herein.

In some embodiments, a polynucleotide cassette of the invention further comprises an RNA export signal. RNA export signals are cis-acting post-transcriptional regulatory elements that enhance the export of RNA from the nucleus. Exemplary RNA export sequences include, but are not limited to, sequences from The hepatitis B Virus post-transcriptional regulatory element (HPRE) and The woodchuck hepatitis Virus post-transcriptional element (WPRE) (Higashimoto, T et al, "woodchuck hepatitis Virus post-transcriptional regulatory element reduces read-through transcription from retroviral vectors," Gene therapy (Gene Ther., 2007, 9 months, 14(17): 1298) -1304).

In some embodiments, a polynucleotide cassette of the invention further comprises a polyadenylation region. As understood in the art, RNA polymerase II transcripts terminate by cleavage and addition of a polyadenylation region, which may also be referred to as the poly (a) signal, poly (a) region, or poly (a) tail. The polyA region contains multiple consecutive adenosine monophosphate, which typically has a repeat of the motif AAUAAA. Several effective polyadenylation sites have been identified, including those from SV40, bovine growth hormone, human growth hormone, and rabbit beta globin (Xu et al, 2001; "Gene (Gene) 272(1-2): 149-. The most effective polyA signal for expression of a transgene in a mammalian cell may depend on the cell type and species of interest and the particular vector used. In some embodiments of the invention, the polynucleotide cassette comprises a polyA region selected from the group consisting of: bovine Growth Hormone (BGH), Human Growth Hormone (HGH), and beta-globin (beta globin).

As understood by one of ordinary skill, two or more of the above-mentioned polynucleotide elements can be combined to produce the polynucleotide cassettes of the present disclosure. Thus, for example, a subject polynucleotide cassette may comprise, in 5' to 3' order, in operable linkage, a CMV enhancer, CMV or EF1 α promoter, optionally a CMVc or EF1 α intron, UTR1, UTR2 or TPL and eMLP 5' UTR, the coding sequence or secreted polypeptide of sFLT1, the entire EES, 410-564EES or 511-810EES enhancer, optionally a HPRE or WPRE RNA export sequence, and a BGH, HGH or β globin polyadenylation signal sequence.

Another polynucleotide cassette may include, in 5' to 3' order, a CMV enhancer, a CMV promoter, a 5' UTR including a TPL sequence and an eMLP sequence, a coding sequence encoding a therapeutic agent (e.g., a therapeutic polypeptide), a full-length EES enhancer, and an HGH polyA signal sequence in operable linkage. In particular embodiments, the coding sequence encodes an anti-angiogenic polypeptide. In particular embodiments, the coding sequence is codon optimized. In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 76-80.

In yet another embodiment, the polynucleotide cassette may comprise, in 5' to 3' order, a CMV enhancer, a CMV promoter, a 5' UTR comprising a TPL sequence and an eMLP sequence, a coding sequence encoding a therapeutic agent, the 410-564EES enhancer, a HPRE RNA export region, and a BGH polyadenylation signal in operable linkage. In particular embodiments, the coding sequence encodes an anti-angiogenic polypeptide. In particular embodiments, the coding sequence is codon optimized. In certain of these embodiments, the polynucleotide cassette includes one or more sequences selected from SEQ ID NOS 81-86.

In yet another example, the polynucleotide cassette may comprise, in 5' to 3' order, in operable linkage, a CMV enhancer, an EF 1a promoter, an EF 1a intron, an UTR 25 ' UTR, a coding sequence encoding a therapeutic agent, a 511-810EES enhancer, a WPRE RNA export region, and a Bovine Growth Hormone (BGH) polyadenylation signal. In particular embodiments, the coding sequence encodes an anti-angiogenic polypeptide. In particular embodiments, the coding sequence is codon optimized. In certain of these embodiments, the polynucleotide cassette comprises one or more sequences selected from SEQ ID NOS 70-75.

In yet another example, the polynucleotide cassette may include, in 5' to 3' order, in operable linkage, a CMV enhancer, a CMV promoter, a CMVc intron, UTR 15 ' UTR, a coding sequence encoding a therapeutic agent, a full length EES enhancer, a WPRE RNA export region, and a beta-globin (beta globin) polyadenylation signal. In particular embodiments, the coding sequence encodes an anti-angiogenic polypeptide. In particular embodiments, the coding sequence is codon optimized. In certain of these embodiments, the polynucleotide cassette includes one or more sequences selected from SEQ ID NOS 92-95.

In yet another example, the polynucleotide cassette may include, in 5 'to 3' order, in operable linkage, a CMV enhancer, an actin promoter, an eMLP intron, a coding sequence encoding a therapeutic agent, a 511-. In particular embodiments, the coding sequence encodes an anti-angiogenic polypeptide. In particular embodiments, the coding sequence is codon optimized. In certain of these embodiments, the polynucleotide cassette includes one or more sequences selected from SEQ ID NOS 87-91.

In yet another example, the polynucleotide cassette may comprise, in 5' to 3' order, in operable linkage, a CMV enhancer, an actin promoter, a chicken β -actin intron, UTR 15 ' UTR, a coding sequence encoding a therapeutic agent, a 410-564EES sequence, an HPRE RNA export sequence, and a BGH polyadenylation signal. In particular embodiments, the coding sequence encodes an anti-angiogenic polypeptide. In particular embodiments, the coding sequence is codon optimized. In certain of these embodiments, the polynucleotide cassette includes one or more sequences selected from SEQ ID NOS 92-95.

As one of ordinary skill in the art will recognize, the polynucleotide cassette may optionally contain other elements, including but not limited to restriction sites to facilitate cloning and regulatory elements for the particular gene expression vector. Examples of regulatory sequences include ITRs of AAV vectors, bacterial sequences of plasmid vectors, attP sites or attB sites of phage integrase vectors, and transposable elements of transposons.

As disclosed herein, in some aspects of the invention, the subject polynucleotide cassettes are used to deliver genes to animal cells, e.g., to determine the effect of the genes on cell viability and/or function, to treat cellular disorders, and the like. Thus, in some aspects of the invention, compositions for expressing a transgene in a mammalian cell are provided as gene delivery vectors, wherein the gene delivery vectors include a polynucleotide cassette of the present disclosure.

The gene delivery vectors of the present disclosure encompass any convenient gene delivery vector for delivering a polynucleotide sequence to a mammalian cell. For example, the vector may comprise single-stranded nucleic acid or double-stranded nucleic acid, such as single-stranded DNA or double-stranded DNA. For example, the gene delivery vector can be DNA, e.g., naked DNA, such as a plasmid or minicircle, and the like. The vector may comprise single-stranded RNA or double-stranded RNA, including modified forms of RNA. In another example, the gene delivery vector may be an RNA, such as an mRNA or a modified mRNA.

As another example, the gene delivery vector may be a viral vector derived from a virus, such as adenovirus, adeno-associated virus (AAV), lentivirus, herpes virus, alphavirus or retrovirus, such as Moloney (Moloney) murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), havey (Harvey) murine sarcoma virus (hamsv), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), Feline Leukemia Virus (FLV), foamy virus, Friend (Friend) murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous (Rous) sarcoma virus (RSV) or lentivirus. Although the examples encompassing the use of adeno-associated viruses are described in more detail below, it is contemplated that one of ordinary skill will recognize that similar knowledge and skills in the art may also be applied to non-AAV gene delivery vectors. See, e.g., the discussion of retroviral vectors in, e.g., U.S. patent No. 7,585,676 and U.S. patent No. 8,900,858, and adenoviral vectors in, e.g., U.S. patent No. 7,858,367, the entire disclosures of which are incorporated herein by reference.

In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In such embodiments, the subject polynucleotide cassettes are flanked at the 5 'and 3' ends by functional AAV Inverted Terminal Repeat (ITR) sequences. By "functional AAV ITR sequence" is meant an ITR sequence that is used as intended to rescue, replicate and package AAV virions. Thus, the AAV ITRs for use in the gene delivery vectors of the invention need not have a wild-type nucleotide sequence and may be altered by insertion, deletion or substitution of nucleotides, or the AAV ITRs may be derived from any of several AAV serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10. Preferred AAV vectors have all or part of the wild-type Rep and Cap genes deleted, but retain functional flanking ITR sequences. In particular embodiments, the AAV viral vector is AAV2 variant 7m8.

In some embodiments, the subject polynucleotide cassette is encapsidated within an AAV capsid, which may be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., any of which may serve as a gene delivery vector. For example, the AAV capsid may be a wild-type capsid or a native capsid. Wild type AAV capsids of particular interest comprise AAV2, AAV5, and AAV 9. However, as with the ITRs, the capsid need not have a wild-type nucleotide sequence, but rather can be altered relative to the wild-type sequence by insertion, deletion or substitution of nucleotides in the VP1, VP2 or VP3 sequences so long as the capsid is capable of transducing mammalian cells. In other words, the AAV capsid may be a variant AAV capsid comprising one or more amino acid substitutions, deletions, or insertions relative to the parental capsid protein or AAV capsid protein from which it is derived. Variant AAVs of particular interest include those disclosed in U.S. patent 9,193,956, the entire disclosure of which is incorporated herein by reference. In some embodiments, the variant AAV comprises a 7m8 variant capsid protein (which may be referred to herein as aav2.7m8 or 7m8.aav2) disclosed in U.S. patent 9,193,956. In other embodiments, the AAV comprises or consists of the AAV2.5T capsid protein provided as SEQ ID No. 42 in U.S. patent No. 9,233,131. In certain embodiments, the AAV comprises an AAVShH10 capsid protein or an AAV6 capsid protein. Figures 8A-8C of U.S. patent application publication No. 20120164106 show the amino acid sequence of the AAVShH10 capsid protein, which is also described in the following: klimczak, R.R. et al, public science library journal (PLOS One) 4(10) e7467 (10/14/2009).

Preferably, the rAAV is replication-defective, in that the AAV vector is unable to independently replicate and package its genome further. For example, when cones are transduced with rAAV virions, the gene is expressed in the transduced cones, however, rAAV cannot replicate due to the fact that the transduced cones lack AAV rep and cap genes as well as helper functions.

Standard methods can be used to generate gene delivery vectors (e.g., rAAV virions) that encapsidate the polynucleotide cassettes of the disclosure. For example, in the case of rAAV virions, an AAV expression vector according to the present invention can be introduced into a producer cell, followed by introduction of an AAV helper construct, wherein the helper construct comprises an AAV coding region capable of expression in the producer cell and which AAV coding region can complement AAV helper functions not present in the AAV vector. A helper virus and/or additional vector is then introduced into the producer cell, wherein the helper virus and/or additional vector provides helper functions capable of supporting efficient rAAV virus production. The producer cells are then cultured to produce rAAV. These steps are performed using standard methods. AAV packaging cells and packaging techniques are used to prepare replication-defective AAV virions that encapsidate the recombinant AAV vectors of the invention by standard techniques known in the art. Examples of these methods can be found, for example, in: U.S. patent No. 5,436,146; U.S. patent No. 5,753,500, U.S. patent No. 6,040,183, U.S. patent No. 6,093,570, and U.S. patent No. 6,548,286, which are expressly incorporated herein by reference in their entirety. Wang et al (US 2002/0168342) describe additional compositions and methods for packaging, which are also incorporated herein by reference in their entirety.

Any concentration of viral particles suitable for efficient transduction of mammalian cells can be prepared for contacting mammalian cells in vitro or in vivo. For example, the viral particles may be formulated at a concentration of 108 or more vector genomes per milliliter (vg/mL), e.g., 5x 108 vector genomes per milliliter; 109 vector genomes per ml; 5 × 109 vector genomes per ml, 1010 vector genomes per ml, 5 × 1010 vector genomes per ml; 1011 vector genomes per ml; 5 × 1011 vector genomes per ml; 1012 vector genomes per ml; 5 × 1012 vector genomes per ml; 1013 vector genomes per ml; 1.5 × 1013 vector genomes per ml; 3 × 1013 vector genomes per ml; 5 × 1013 vector genomes per ml; 7.5 × 1013 vector genomes per ml; 9 × 1013 vector genomes per ml; 1X 1014 vector genomes per ml, 5X 1014 or more vector genomes per ml, but generally not more than 1X 1015 vector genomes per ml. Similarly, any total number of viral particles suitable to provide appropriate cell transduction to confer a desired effect or treat a disease may be administered to a mammal. In various preferred embodiments, at least 108; 5 × 108; 109 are provided; 5 × 109, 1010, 5 × 1010; 1011; 5 × 1011; 1012; 5 is multiplied by 1012; 1013 pieces of; 1.5X 1013 pieces; 3 × 1013 pieces; 5 × 1013 pieces; 7.5 × 1013 pieces; 9 × 1013, 1 × 1014 or 5 × 1014 or more virus particles, but usually not more than 1 × 1015 virus particles are injected per eye. Any suitable number of vectors can be administered to the mammalian or primate eye. In one embodiment, the method comprises a single administration; in other embodiments, multiple administrations may be performed over time as deemed appropriate by the attending clinician.

the subject viral vectors can be formulated into pharmaceutical compositions comprising any suitable unit dose of the vector, which can be administered to a subject to produce a change in the subject or to treat a disease in the subject. In some embodiments, a unit dose includes, but is not limited to, 1 × 108 or more vector genomes of a viral vector, e.g., at least about 1 × 109, 1 × 1010, 1 × 1011, 1 × 1012, 1 × 1013, 1 × 1014, or at least about 3 × 1014 or more vector genomes, in some cases, at least about 1 × 1014 vector genomes, but typically no more than 4 × 1015 vector genomes. In some cases, a unit dose includes up to about 5 × 1015 vector genomes, e.g., 1 × 1014 or 5 × 1014 or less vector genomes, e.g., 1 × 1013, 1 × 1012, 1 × 1011, 1 × 1010, or 1 × 109 or less vector genomes, in some cases 1 × 108 or less vector genomes, and typically no less than 1 × 108 vector genomes. In some cases, a unit dose comprises 1 × 1010 to 1 × 1011 vector genomes. In some cases, a unit dose comprises 1 × 1010 to 3 × 1012 vector genomes. In some cases, a unit dose comprises 1 × 109 to 3 × 1013 vector genomes. In some cases, a unit dose comprises 1 × 108 to 3 × 1014 vector genomes. In some cases, a unit dose comprises about 1 × 1010 to about 5 × 1014 vector genomes.

In some cases, the multiplicity of infection (MOI) can be used to measure the unit dose of a pharmaceutical composition. MOI refers to the ratio or fold of vector or viral genome to cells into which nucleic acid can be delivered. In some cases, the MOI may be 1 × 106. In some cases, the MOI may be 1 × 105 to 1 × 107. In some cases, the MOI may be 1 × 104 to 1 × 108. In some cases, the recombinant viruses of the present disclosure are at least about 1 × 101, 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107, 1 × 108, 1 × 109, 1 × 1010, 1 × 1011, 1 × 1012, 1 × 1013, 1 × 1014, 1 × 1015, 1 × 1016, 1 × 1017, and 1 × 1018 MOIs. In some cases, the recombinant viruses of the present disclosure are 1 × 108 to 3 × 1014 MOIs. In some cases, the recombinant viruses of the present disclosure are up to about 1 × 101, 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107, 1 × 108, 1 × 109, 1 × 1010, 1 × 1011, 1 × 1012, 1 × 1013, 1 × 1014, 1 × 1015, 1 × 1016, 1 × 1017, and 1 × 1018 MOIs.

in some aspects, the amount of the pharmaceutical composition comprises from about 1 x 108 to about 1 x 1015 recombinant viruses, from about 1 x 109 to about 1 x 1014 recombinant viruses, from about 1 x 1010 to about 1 x 1013 recombinant viruses, or from about 1 x 1011 to about 3 x 1012 recombinant viruses.

In preparing the subject rAAV compositions, any host cell for producing rAAV virions can be employed, including, but not limited to, e.g., mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell may also be a packaging cell in which the AAV rep and cap genes are stably maintained in the host cell or a producer cell in which the AAV vector genome is stably maintained and packaged. Exemplary packaging and production cells are derived from SF-9, 293, A549, or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

The invention encompasses pharmaceutical compositions comprising a polynucleotide cassette or gene delivery vector as described herein and a pharmaceutically acceptable carrier, diluent or excipient. For example, one embodiment is a pharmaceutical composition comprising a recombinant virus comprising a polynucleotide of the present disclosure and a pharmaceutically acceptable excipient. In particular embodiments, the recombinant virus is a recombinant adeno-associated virus (AAV). The subject polynucleotide cassettes or gene delivery vectors can be combined with pharmaceutically acceptable carriers, diluents, and agents that can be used to prepare formulations that are generally safe, non-toxic, and desirable and that contain acceptable excipients for primate use. Such excipients may be solid, liquid, semi-solid or gaseous (in the case of aerosol compositions). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds may also be incorporated into these formulations. The solution or suspension for the formulation may comprise: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; detergents, such as Tween (Tween)20 to prevent aggregation; and compounds for regulating tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). In particular embodiments, the pharmaceutical composition is sterile.

For the case of cone cell contact in vivo, the subject polynucleotide cassette or a gene delivery vector comprising the subject polynucleotide cassette may be considered suitable for delivery to the eye.

Pharmaceutical compositions suitable for use in the present invention further comprise sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Thus, the pharmaceutical compositions may be in the form of sterile injectable solutions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or Phosphate Buffered Saline (PBS). In some cases, the composition is sterile and should have fluidity to the extent that it is easy to inject. In certain embodiments, the compositions are stable under the conditions of manufacture and storage, and are preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the internal composition can be brought about by including in the composition an agent that delays absorption (e.g., aluminum monostearate and gelatin).

if desired, sterile solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the composition is prepared with a carrier that will protect the gene cassette or expression vector from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available. Liposomal suspensions (comprising liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These formulations can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is particularly advantageous to formulate oral, ophthalmic or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined amount of the gene delivery vector or polynucleotide cassette calculated to produce the desired therapeutic effect in association with the desired drug carrier. The specification for the dosage unit form of the invention is dictated by the unique characteristics of the gene delivery vector, polynucleotide cassette and the particular therapeutic effect to be achieved.

The pharmaceutical composition can be contained in a container, package, or dispenser (e.g., a syringe, such as a prefilled syringe) with instructions for administration.

By "pharmaceutically acceptable excipient" is meant a material, substance, diluent or carrier that is substantially non-toxic to the cells or subject to which it is to be administered. That is, a pharmaceutically acceptable excipient may be incorporated into a pharmaceutical composition and administered to a cell or patient without substantially causing an undesirable biological effect or interacting in a deleterious manner with any of the other components of the composition in which the pharmaceutically acceptable excipient is contained.

The subject polynucleotide cassettes or gene delivery vectors (e.g., recombinant viruses (virions)) can be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly primates and more particularly humans. The subject polynucleotide cassettes or gene delivery vectors (e.g., viral particles) can be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier having a pH preferably in the range of 3 to 8, more preferably in the range of 6 to 8, or even more preferably in the range of 7 to 8. Such sterile compositions will include vectors or viral particles containing a nucleic acid encoding a therapeutic molecule dissolved in an aqueous buffer having an acceptable pH upon reconstitution.

In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of the carrier or viral particle in admixture with a pharmaceutically acceptable carrier and/or excipient, e.g., saline, phosphate buffered saline, phosphate, and optionally one or more other agents, such as amino acids, polymers, polyols, sugars, buffers, preservatives, proteins, and inorganic salts (e.g., sodium chloride). Exemplary amino acids, polymers, and sugars and the like are octylphenoxy polyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, ringer's and Hank's (Hank's) solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and ethylene glycol. Preferably, such formulations are stable at 4 ℃ for at least six months.

In some embodiments, the pharmaceutical compositions provided herein include buffers such as Phosphate Buffered Saline (PBS) or sodium phosphate/sulfate, tris buffers, glycine buffers, sterile water, and other buffers known to those of ordinary skill, such as those described by Good et al, (1966) Biochemistry 5(2) 467-477. The pH of the buffer may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4. The pharmaceutical compositions can include an adenovirus or adeno-associated adenovirus vector delivery system comprising a polynucleotide cassette of the present disclosure.

Method of producing a composite material

The ability to deliver the gene expression cassettes of the invention to selected target cells in vivo and to obtain a therapeutically effective amount of the gene product in the cells and in the extracellular environment following a transduction event may be beneficial for the treatment of a number of different diseases, including those that are dependent on the growth of new blood vessels, where the goal of treatment may be the ability of the target cells to secrete a therapeutically effective amount of an anti-angiogenic protein. While not wishing to be bound by any theory, even when the gene delivery vector is able to successfully transduce only a fraction of the target cells, an expression cassette that is capable of enhancing expression and ultimately therapeutic protein secretion may help provide a clinically significant benefit to the patient. High levels of secretion of therapeutic proteins by transduced cells may help balance the infectivity or transduction efficiency achieved with any given dose or gene therapy round.

Thus, the subject polynucleotide cassettes and gene delivery vectors, collectively referred to herein as "subject compositions," can be used to express transgenes in animal cells. For example, the subject compositions can be used to study, e.g., determine the effect of a gene on cell viability and/or function. As another example, the subject compositions may be used in medicine, for example, to treat a disorder. The methods and compositions of the present disclosure may be used to treat any condition that can be at least partially addressed by gene therapy of cells. Cells include, but are not limited to, blood, eye, liver, kidney, heart, muscle, stomach, intestine, pancreas, and skin.

Accordingly, the present invention provides a method for treating or preventing a disease or disorder (e.g., an ocular disease or disorder) in a subject in need thereof, the method comprising administering to the subject in need thereof a viral vector or viral particle comprising a polynucleotide cassette of the invention encoding a therapeutic gene product. In a preferred embodiment, the therapeutic gene product is a secreted polypeptide or a protein that is secreted or exported from the cell after synthesis in the cell, and the polynucleotide cassette comprises, in 5 'to 3' order: (a) a first enhancer region comprising a CMV sequence (SEQ ID NO: 1); (b) a promoter region comprising a CMV sequence (SEQ ID NO: 4); (c) a 5' UTR region comprising, in 5' to 3' order, a TPL sequence and an eMLP sequence (SEQ ID NO:11 and SEQ ID NO:12, respectively); (d) a coding sequence encoding a peptide or polypeptide; (e) a second enhancer region comprising the entire EES sequence (SEQ ID NO: 13); and (f) an HGH polyadenylation site (SEQ ID NO: 14).

In related embodiments, some methods provide for expression of a gene in a cell in vitro or in vivo, the method comprising contacting the cell with a composition of the disclosure. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to a subject. The composition may be administered parenterally by intravenous injection or oral infusion. In certain embodiments, the composition is administered to the eye by injection, for example to the retina, lower retina, or vitreous. In certain embodiments, the composition is administered by retinal injection, subretinal injection, or intravitreal injection. In certain embodiments, the composition is administered topically or directly to a tissue or organ of interest, for example by injection into the liver.

The subject can be a mammal, including, for example, a human subject in need of treatment for a particular disease or condition.

For the case where mammalian cells are contacted with the subject polynucleotide cassette or gene delivery vector comprising the subject polynucleotide cassette in vitro, the cells can be from any mammalian species, e.g., rodent (e.g., mouse, rat, gerbil, squirrel), rabbit, cat, dog, goat, sheep, pig, horse, cow, primate, human. The cells may be from an established cell line or may be primary cells, wherein "primary cells", "primary cell lines" and "primary cultures" are used interchangeably herein to refer to cells and cell cultures derived from a subject and allow for a limited number of passages, i.e., divisions, of a growing culture in vitro. For example, a primary culture is a culture that may have been passaged 0,1, 2, 4,5, 10, or 15 times, but not enough to undergo a crisis stage. Typically, the primary cell lines of the invention are maintained in vitro for less than 10 passages.

if the cells are primary cells, the cells may be harvested from the mammal by any convenient method, e.g., whole explants, biopsies, etc. The collected cells may be dispersed or suspended with a suitable solution. Such a solution will typically be a balanced salt solution, such as physiological saline, PBS, hank's balanced salt solution, or the like, conveniently supplemented with fetal bovine serum or other naturally occurring factors at low concentrations (typically 5-25mM) in combination with an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. The cells may be used immediately or may be stored, frozen for extended periods of time, thawed and capable of being reused. In this case, cells are typically frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art, to preserve the cells at such freezing temperatures and to be thawed in a manner as is commonly known in the art for thawing frozen cultured cells.

To facilitate expression of the transgene, the subject polynucleotide cassette or a gene delivery vector comprising the subject polynucleotide cassette is contacted with the cell for about 30 minutes to 24 hours or more, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, 24 hours, etc. The subject polynucleotide cassette or gene delivery vector comprising the subject polynucleotide cassette can be provided to the subject cells one or more times (e.g., once, twice, three times, or more than three times) and the cells are allowed to incubate with the agent(s) for a time after each contact event (e.g., 16-24 hours), after which the medium is replaced with fresh medium and the cells are further cultured. Contacting the cells can occur in any medium and under any culture conditions that promote cell survival. For example, the cells may be suspended in any suitable convenient nutrient medium, such as Iscove's modified DMEM or RPMI 1640, supplemented with foetal calf serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, especially 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. As defined herein, a growth factor is a molecule capable of promoting the survival, growth and/or differentiation of cells in culture or in intact tissues through specific action on transmembrane receptors. Growth factors include polypeptide factors and non-polypeptide factors.

Generally, an effective amount of the subject polynucleotide cassette or a gene delivery vector comprising the subject polynucleotide cassette is provided to produce transgene expression in a cell. As discussed elsewhere herein, an effective amount can be readily determined empirically, e.g., by detecting the presence or level of a transgene product, by detecting an effect on cell viability or function, etc. Typically, expression will be enhanced 2-fold or more, e.g., 3-fold, 4-fold, or 5-fold or more, in some cases 10-fold, 20-fold, or 50-fold or more, e.g., 100-fold, relative to expression by a reference or control polynucleotide cassette. One example of a reference cassette for comparison purposes is the CMV reference control cassette described herein. In particular embodiments, the transgene encodes a secreted protein, and the polynucleotide cassette expresses the secreted protein in the mammalian cell at a level that is at least 2-fold, 5-fold, 10-fold, 5-fold to 15-fold, or 10-fold to 15-fold greater than the level of expression of the secreted protein in the mammalian cell by the CMV reference control cassette. According to some embodiments, when the transgene is a transgene encoding a non-secretory protein, the polynucleotide cassette of the invention expresses the non-secretory protein in the mammalian cell at a level that is about the same as, within 10-20% of, about 1.5-fold lower than, or about 2-fold lower than the level of expression of the non-secretory protein in the mammalian cell by the CMV reference control cassette. The expression level of secreted protein for each cassette can be measured by an immunoassay or an antigen capture assay, and can be expressed as the amount or concentration of protein per volume of supernatant (e.g., cell culture medium or supernatant) in the extracellular environment.

Immunoassays for measuring The presence and amount (and thus The expression level) of proteins in biological or cellular samples are known in The art (e.g., Hage, d.s. (1999) "Immunoassays" (Analytical Chemistry), "71 (12) in Analytical Chemistry 294-. Typically, immunoassays are based on the reaction between a target protein and an antibody or antibody fragment that specifically binds to the protein. Immunoassays can be carried out in liquid phase or solid phase systems, but for ease of detection, a solid phase may be preferred. Suitable immunoassays include, but are not limited to, sandwich and competition assays, western blots, ELISA (enzyme linked immunosorbent assays), Radioimmunoassays (RIA), Fluorescent Immunoassays (FIA), and the like. The biological sample may be cell culture medium or supernatant (a sample taken from a culture without lysing the cells), cell lysate, whole cells, blood, serum, plasma, or other bodily fluids or tissues. In some embodiments, such as when the transgene is a selectable marker, the cell population can be enriched for those comprising the subject polynucleotide cassette by separating the modified cells from the remaining population. The separation may be carried out by any convenient separation technique appropriate for the selectable marker used. For example, if the transgene is a fluorescent label, the cells may be isolated by fluorescence activated cell sorting, whereas if the transgene is a cell surface label, the cells may be separated from the heterogeneous population by affinity separation techniques, such as magnetic separation, affinity chromatography, "panning" with affinity reagents attached to a solid matrix, or other convenient techniques. Techniques for providing accurate separation include fluorescence activated cell sorters, which may have varying degrees of complexity, such as multi-color channels, low and obtuse angle light scatter detection channels, impedance channels, and the like. Cells can be selected for dead cells by using a dye (e.g., propidium iodide) associated with the dead cells. Any technique that is not unduly detrimental to cell viability may be employed. In this way, a highly enriched cell composition of cells comprising the subject polynucleotides is achieved. By "highly enriched" is meant that the genetically modified cells will be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the cellular composition, for example about 95% or more or 98% or more of the cellular composition.

For the case where the cell is contacted with the subject polynucleotide cassette or a gene delivery vector comprising the subject polynucleotide cassette in vivo, the subject can be any mammal, e.g., a rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow, human, or non-human primate.

the methods and compositions of the invention may be used to treat any condition that can be at least partially addressed by gene therapy of cells. Cells include, but are not limited to, blood, eye, liver, kidney, heart, muscle, stomach, intestine, pancreas, and skin. One embodiment is a method for treating a medical condition in a subject in need of treatment, the method comprising administering to the subject a gene delivery vector comprising a polynucleotide cassette as disclosed herein, wherein the cassette encodes a polypeptide effective to reduce one or more signs or symptoms of the medical condition. In some embodiments, the medical condition is an ocular disease, the gene delivery vector is an adeno-associated virus, and the polypeptide is a polypeptide secreted by a cell transduced by the vector. In one embodiment, the secreted protein inhibits VEGF signaling. For example, the secreted protein may be a VEGF-binding protein. In some embodiments, the ocular disease is choroidal neovascularization or macular degeneration. Particular forms of macular degeneration may include acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration. Administration may be by any suitable means, including, for example, ocular delivery, intravitreal injection, intraocular injection, retinal injection, subretinal injection, parenteral administration, intravenous injection or infusion, and injection into the liver.

In some embodiments, the gene delivery vector is administered to the eye of a subject in need of treatment. In some embodiments, the gene delivery vector is administered to the subject by intraocular injection, by intravitreal injection, or by any other convenient mode or route of administration. In some embodiments, the subject is a human subject having or at risk of developing macular degeneration or ocular neovascularization.

In some embodiments, the subject methods result in therapeutic benefit, such as preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, and the like. In some embodiments, the subject methods include the step of detecting whether a therapeutic benefit is achieved. The ordinarily skilled artisan will understand that such measurement of treatment efficacy will be appropriate for the particular disease being modified, and will recognize appropriate detection methods for measuring treatment efficacy.

It is desirable that the transgene expression obtained using the subject transgenes be robust. Thus, in some cases, transgene expression (e.g., as detected by measuring levels of gene product) can be observed two months or less after administration (e.g., 4 weeks, 3 weeks, or 2 weeks or less after administration (e.g., 1 week after administration of the subject composition)) by measuring therapeutic efficacy, or the like. It is expected that transgene expression will also persist over time. Thus, in some cases, transgene expression (e.g., as detected by measuring levels of a gene product) can be observed 2 months or more (e.g., 4 months, 6 months, 8 months, or 10 months or more, in some cases, 1 year or more, e.g., 2 years, 3 years, 4 years, or 5 years, in some cases more than 5 years) after administration of the subject compositions by measuring therapeutic efficacy, or the like.

In certain embodiments, the methods comprise the step of detecting transgene expression in the cell, wherein expression is enhanced relative to expression by a polynucleotide cassette (i.e., a reference control) that does not include one or more improved elements of the present disclosure. Typically, expression will be enhanced 2-fold or more, e.g., 3-fold, 4-fold or 5-fold or more, in some cases 10-fold, 20-fold or 50-fold or more (e.g., 100-fold) relative to expression by the reference (i.e., control polynucleotide cassette), as evidenced by, e.g., early detection, higher levels of gene product, greater functional impact on the cell, etc. In one aspect, the transgene encodes a secreted polypeptide (e.g., sFLT 1).

Typically, if the subject composition is a virus (e.g., a rAAV comprising a polynucleotide cassette of the present disclosure), an effective amount for achieving a change or producing a therapeutic effect in a subject will be about 1 × 108 vector genomes or more, in some cases, 1 × 109, 1 × 1010, 1 × 1011, 1 × 1012, or 1 × 1013 vector genomes or more, in some cases, 1 × 1014 vector genomes or more, and typically no more than 1 × 1016 vector genomes. In some cases, the amount of vector genome delivered is up to about 1 × 1016 vector genomes, e.g., 1 × 1015 vector genomes or less, e.g., 1 × 1013, 1 × 1012, 1 × 1011, 1 × 1010, or 1 × 109 vector genomes or less, in some cases, 1 × 108 vector genomes, and typically no less than 1 × 108 vector genomes. In some cases, the amount of vector genome delivered is 1 × 1010 to 1 × 1011 vector genomes. In some cases, the amount of vector genomes delivered is 1 x 1010 to 3 x 1012 vector genomes. In some cases, the amount of vector genome delivered is 1 × 109 to 3 × 1013 vector genomes. In some cases, the amount of vector genomes delivered is 1 × 108 to 3 × 1014 vector genomes.

In some cases, the multiplicity of infection (MOI) can be used to measure the amount of the pharmaceutical composition to be administered. In some cases, MOI may refer to the ratio or fold of vector particle or viral genome to cells to which the polynucleotide cassette may be delivered. In some cases, the MOI may be 1 × 106. In some cases, the MOI may be 1 × 105 to 1 × 107. In some cases, the MOI may be 1 × 104 to 1 × 108. In some cases, the recombinant viruses of the present disclosure are about 1 × 101, 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 MOIs.

In some aspects, the pharmaceutical composition comprises from about 1 x 108 to about 1 x 1015 recombinant viral particles, from about 1 x 109 to about 1 x 1014 recombinant viral particles, from about 1 x 1010 to about 1 x 1013 recombinant viral particles, or from about 1 x 1011 to about 3 x 1012 recombinant viral particles.

The individual dose is generally no less than the amount required to produce a measurable effect on the subject, and can be determined based on the pharmacokinetics and pharmacology of absorption, distribution, metabolism, and excretion ("ADME") of the subject composition or its byproducts, and thus, the disposition of the composition within the subject. This includes consideration of the route of administration and dosage. Effective dosages and/or dosage regimens can be readily determined empirically, by preclinical determination, by safety and escalation and dose ranging trials, by individual clinician-patient relationships, and by in vitro and in vivo assays.

All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification are incorporated herein by reference in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

examples of the invention

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Although efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric pressure.

general methods in molecular and cellular biochemistry can be found in such standard textbooks as "molecular cloning: a Laboratory Manual, 3 rd edition (Sambrook et al, Cold Spring Harbor Laboratory Press 2001); finely compiled Molecular Biology Protocols in Molecular Biology, 4 th edition (edited by Ausubel et al, John Wiley & Sons 1999); protein Methods (Protein Methods) in the book (Bollag et al, John Willi, parent-Press 1996); non-viral Vectors for Gene Therapy (non viral Vectors for Gene Therapy) (edited by Wagner et al, Academic Press 1999); viral vectors (Viral Vector) (Kaplift and Loewy, ed., academic Press 1995); manual of immunological Methods (Immunology Methods Manual) (edited by i.e. lefkovits, academic press 1997); and "cell and tissue culture: biotechnology Laboratory programs (Cell and Tissue Culture: Laboratory Procedures in Biotechnology) (Doyle and Griffiths, John Willi, parent-child publishing company 1998), the disclosure of which is incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are commercially available from commercial suppliers, such as BioRad, Stratagene, Invitrogen, Sigma Aldrich (Sigma-Aldrich), and ClonTech.

example 1

Construction of the Polynucleotide expression cassette

Important considerations in the development of any gene therapy approach are the vehicle used to deliver the gene and the expression cassette used to drive transgene production once inside the cell. The cassette is preferably one that promotes robust expression of the transgene at a level sufficient to provide rapid and sustained therapeutic benefit to the patient. To this end, a series of polynucleotide expression cassettes containing various combinations and permutations of regulatory elements and protein coding sequences were generated using standard recombinant DNA cloning techniques (FIG. 1).

example 2

Construction of recombinant plasmid

Recombinant plasmids comprising each of the candidate polynucleotide cassettes and the coding sequence encoding aflibercept were constructed and cloned in e.coli using conventional DNA recombination and cloning techniques. As shown in the vector diagram of cassette 11 (fig. 11), each cassette was positioned between Inverted Terminal Repeats (ITRs) of adeno-associated virus serotype 2(AAV2) for subsequent transfer of the cassette into the AAV genome and production of recombinant AAV virions.

As shown in fig. 11, cassette 11 comprises, in 5' to 3' order, a CMV enhancer, a CMV promoter, a 5' UTR including sequences from TPL and eMLP, a protein coding sequence, a complete Expression Enhancer Sequence (EES), and a human growth hormone polyadenylation signal sequence (HGH polyA). In a similar manner, cassette 12 comprises, in 5' to 3' order, a CMV enhancer, CMV promoter, TPL and eMLP 5' UTR sequences, protein coding sequences, the 410-564 portion of the expression enhancer sequence (410-564EES), cis-acting post-transcriptional regulatory elements of hepatitis B virus (HPRE) and a bovine growth hormone polyadenylation signal sequence (BGH polyA). As used in these studies, the polynucleotide sequences of cassettes 10, 11, 12 and 14 and the CMV reference control cassette are shown in tables 1, 2,3, 4 and 5, respectively. Starting from the top of the table and continuing to the bottom of the table, the ITRs, regulatory elements and coding regions are listed as they appear in the plasmid in 5' to 3 order. A CMV reference cassette was also constructed (table 5) and tested in side-by-side comparison with other selected cassettes of the present disclosure.

Example 3

Protein expression in vitro transfected mammalian cells

For in vitro evaluation of the expression profile of each cassette, each recombinant construct was transfected into mammalian cells using 6 transfection reagents (Promega). In the first series of experiments (fig. 2), the cassette encodes proteins secreted from the cells upon translation. After transfection, cells were incubated for 48 hours. Cell culture supernatants from each culture were then sampled and assayed by immunoassay to assess the level of secreted protein by each transfected cell culture. FIG. 2 shows the expression levels of the cassettes C1-C18 relative to the expression levels of the "base plasmid" cassette depicted in FIG. 1. As shown in figure 2, the highest average expression of HeLa cells was observed for those cells transfected with cassettes 11 and 12.

Example 4

Protein expression in vitro transduced mammalian cells

Based on the results shown in fig. 2, five cassettes (C7, C11, C12, C13, and C14) were selected for further study. Experiments were performed to compare the expression of secreted proteins by each cassette when delivered by recombinant adeno-associated virus to mammalian cells in vitro. The C7, C11, C12, C13 and C14 cassettes shown in fig. 2 for HeLa cell studies were each packaged within the AAV7m8 capsid (Dalkara et al, "scientific transformation medicine (sci. trans. med.)," 2013, vol 5, stage 189, 189ra 76). Individual cultures of HEK293 cells were transduced with each recombinant AAV7m8 vector at 3 × 105 MOI and then incubated for 3 days. After incubation, supernatants were collected from each culture and assayed using an immunoassay to measure the amount of secreted protein in the supernatant of each culture during the 3 day incubation. As in transfected HeLa cells (fig. 2), the highest expression in HEK293 cells was observed for those transduced with cassettes 11 and 12 (fig. 3).

Example 5

protein expression in transduced porcine retinal explant cultures

The recombinant aav2.7m8 vector shown in figure 3 for study was further tested in a porcine retina explant culture system. Porcine retina has similar anatomical and physiological characteristics as humans and therefore can serve as a suitable substitute in preclinical testing. Whole retina explant cultures retain complex intracellular processes and communication between neural retinal cells and are a useful model in target tissue validation of AAV vector variants.

Transduction of porcine retina explants

Aav2 vectors are variant AAV2 vectors that are capable of better transducing photoreceptors than wild-type AAV2 (Dalkara et al, scientific transformation medicine, "In Vivo Directed Evolution of a New Adeno-Associated Virus for Therapeutic epiretinal Gene Delivery from the Vitreous" (In Vivo-Directed Evolution of a New adono-Associated Virus for Therapeutic Outer Retinal Gene Delivery), "2013, volume 5, stage 189, 189ra 76). Porcine retina explants with whole retina were transduced with 7m8 vector at 2 × 104 MOI. Supernatants were collected one and two weeks after transduction and the amount of secreted protein present in the supernatants was measured by immunoassay. The experiments were performed in duplicate (explants 1 and 2) and figure 4 shows the results showing the levels of protein expressed and secreted from each transduced explant as measured by the amount of protein in the culture supernatant, along with the background levels of the untransduced "vehicle" control explants. As shown in figure 4, the trend in protein expression levels for this set of cassettes correlated with the trend observed for transduced HEK293 cells in vitro (figure 3), with cassettes 11, 12 and 14 providing the three highest expression levels and cassettes 7 and 13 providing the lowest expression level.

Example 6

Expression of SFLT-1 in mammalian cells transfected in vitro

It is of interest to study the expression characteristics of the cassette with respect to other proteins and other mammalian cell types. For this purpose, the coding sequences for two other proteins (sFLT-1 and Green Fluorescent Protein (GFP)) were cloned into polynucleotide cassettes C10, C11 and C12, respectively. For comparison, the sequences encoding sFLT1 were also cloned into the base plasmid cassette (fig. 1) and the CMV control cassette ("CMV-sFLT 1"), respectively. As described in Table 5, the CMV-sFLT1 control construct included, in 5 'to 3' order, a CMV enhancer sequence (SEQ ID NO:2), a CMV promoter (SEQ ID NO:21), a chimeric intron (SEQ ID NO:22), a 5'UTR (SEQ ID NO:23), a coding sequence encoding sFLT-1 (SEQ ID NO:24), a 3' UTR (SEQ ID NO:25), and an SV40polyA sequence (SEQ ID NO: 26). In addition to the CMV-sFLT1 control construct, the coding sequence for sFLT-1 (also referred to herein as sFLT1) was Codon Optimized (CO) for expression in primate cells.

sFLT1 encoding vectors were transfected into three different cell lines: retinal pigment epithelial cell lines (ARPE19 cells), HEK293 cells and HeLa cells. Transfection was performed using 6 transfection reagents. After the transfection step, the cells were incubated for 48 hours and the amount of sFLT1 protein present in the cell culture supernatant was measured using sFLT1ELISA kit from R & D Systems. Plasmids encoding GFP under the control of cassettes 7, 11 or 13 were transfected into HeLa cells and compared to plasmids expressing GFP under the control of a CMV promoter (CMV-sFLT1, as described above). After approximately 48 hours, the cells were trypsinized and the percentage of GFP positive cells in each culture was assessed by flow cytometry (BD facscalibur (tm)). The results are shown in FIGS. 5-8. In general, the highest expression of sFLT1 was observed in cells transfected with cassettes 11 and 12 compared to any of the other cassettes tested (fig. 5-7). More specifically, sFLT1 expression by cassette 11 in ARPE19 cells was about 2.2-fold (or about 2-fold) greater than sFLT1 expression by the CMV-sFLT1 control cassette in ARPE19 cells (fig. 5). The expression of sFLT1 in HEK293 cells by cassette 11 was about 9-fold higher than the expression of sFLT1 in HEK293 cells by the CMV-sFLT1 control cassette (fig. 6). The expression of sFLT1 in transfected HeLa cells from cassette 11 was about 9-fold higher than the expression of sFLT1 in HeLa cell culture from the CMV-sFLT1 control cassette (fig. 7). However, surprisingly, while the level of sFLT1 expressed by cassette 11 was consistently higher than the level of sFLT1 expressed by any of the other tested cassettes and significantly higher than the level of sFLT1 expressed by the CMV-sFLT1 control cassette in each cell line tested (fig. 5-7), cassette 11 provided similar levels of protein per cell when the coding sequence was changed to a sequence encoding GFP compared to the CMV control cassette. As shown in figure 8, the fold difference in the amount of protein expressed in mammalian cells by cassette 11 and CMV-sFLT1 previously observed when each cassette encodes sFLT1 was significantly reduced when the coding sequence changed from a sequence encoding sFLT1 (a secreted protein) to a sequence encoding GFP (a non-secreted cytoplasmic protein). Based on these unexpected results, it is believed that cassette 11 is particularly suitable for expressing polypeptides secreted from cells, as compared to other cassettes.

example 7

sFLT-1 expression in transduced mammalian cells

In a further study, C10 and C11 cassettes containing codon-optimized coding sequences encoding human FLT-1 and a control CMV-sFLT-1 cassette were packaged in 7m8 capsids to form recombinant 7m8.aav2 virions encoding sFLT-1 under the control of the respective cassette constructs. The CMV-sFLT1 control construct was also packaged in a wild-type AAV2 capsid (AAV2-CMV-sFLT 1). HEK293 cells were transduced with each adeno-associated virus construct at 1 × 105 MOI. Three days after transduction, supernatants from each culture were collected and the concentration of sFLT-1 in each supernatant sample was determined using an sFLT-1ELISA kit. As shown in figure 9, the highest expression was observed in HEK293 cells transduced with cassette 11 (C11). The expression of sFLT-1 in transduced HEK293 cells from cassette 11 was approximately 50 times (33661 pg/mL compared to 718 pg/mL) greater than that of sFLT-1 from the control CMV-sFLT1 cassette. The data shown on the right side of fig. 9 shows that further dilution of the supernatant from C11 transduced cells was necessary to bring the sample within the range of the assay standard curve.

example 8

Expression of sFLT-1 in transduced porcine retinal explants

to compare the expression of sFLT-1 retinal tissue, porcine retinal explants were transduced with each of the recombinant AAV vectors shown in fig. 9. Experiments were performed in triplicate and each explant was transduced at 4 × 104 MOI. After transduction, the medium was changed every 3 days. Two weeks later, medium was collected from each explant culture for 3 days and the amount of sFLT-1 from each culture in the medium was assessed using the sFLT-1ELISA kit. In addition, retinal explants were lysed and tissue lysates were assayed by ELISA for intracellular expression of sFLT-1. The results are shown in FIG. 10. As shown in figure 10, the amount of sFLT-1 in the supernatant from the explants transduced with aav2.7m8 encoding sFLT-1 under the control of cassette 11(7m8-C11-co.sflt) was on average about 60 times the amount of sFLT-1 in the supernatant of the explants transduced with aav2.7m8 encoding sFLT-1 under the control of CMV cassette (7m8-CMV-sFLT 1). The relative difference in the amount of sFLT-1 in the supernatants from the various explant cultures correlated with the relative difference in the amount of sFLT-1 protein found intracellularly (fig. 10; tissue lysates), suggesting that cassette 11 promoted higher expression of sFLT-1 in mammalian cells, and as shown in fig. 9 and 10, thereby promoting secretion of higher levels of transduced extracellular protein in extracellular medium compared to the levels of sFLT-1 observed in and around cells transduced with the 7m8-CMV-sFLT1 control cassette and other tested sFLT-1-encoding cassettes (i.e., in the extracellular environment of the cells).

Example 9

Abutip expression in transduced gerbil eyes

In a further study, aflibercept expression driven by sand boxes C7, C11, C12, C13, C14 was compared in gerbils. AAV vectors were assembled as described in example 4. In addition, AAV7m8 capsids containing MNTC expression cassettes expressing codon optimized aflibercept were assembled. A group of 8 animals received a bilateral Intravitreal (IVT) injection at a dose of 2x 1010 vg/eye with either vehicle: AAV.7m8-C7-Co-Abercept, AAV.7m8-C11-Co-Abercept, AAV.7m8-C12-Co-Abercept, AAV.7m8-C13-Co-Abercept or AAV.7m8-C14-Co-Abercept. Four animals were sacrificed at 8 and 16 weeks post-injection, dissected for (i) retina, (ii) vitreous, (iii) retina/choroid, and (iv) iris/ciliary body, and subjected to aflibercept expression analysis for each eye (total eight eyes per time point per group).

Free aflibercept expression in tissue samples was measured using a modified sandwich ELISA. Specifically, microtiter plates were coated with recombinant human VEGF protein. Protein samples were incubated in each well, followed by washing of each well. After washing, horseradish peroxidase (HRP) conjugated to an anti-human IgG monoclonal antibody was added to each well. The antibody binds to the Fc domain of aflibercept protein, which is itself captured by VEGF bound to the surface of the well. After incubation, wells were washed and bound enzyme activity was determined by adding Luminol (Luminol) and then measuring light emission at 466 nm.

Expression was detected from each construct in each of the tissue samples examined at the two mild time points of 8 and 16, and was highest in the vitreous of each construct at the two time points.

As exemplified by cassette 11, Inverted Terminal Repeat (ITR) sequences from AAV may be placed in flanking positions around any of the cassettes for subsequent transfer of the cassettes to the AAV genome. A Kozak sequence (e.g., GCCACC) may occur 5' to the start codon of the coding sequence. The CMV reference control cassette can include any transgene of interest. Table 5 shows an illustrative example in which the transgene encodes sFLT-1.

Sequence listing

<110> Keravala, Annahita

<120> compositions and methods for enhancing gene expression

<130> AVBI-009/00US

<160> 97

<170> PatentIn version 3.5

<210> 1

<211> 293

<212> DNA

<213> Human cytomegalovirus (Human cytomegavirus)

<400> 1

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 60

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 120

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc 180

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 240

acgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta cca 293

<210> 2

<211> 659

<212> DNA

<213> Human cytomegalovirus (Human cytomegavirus)

<400> 2

tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60

ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120

aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360

ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480

gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540

caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600

caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataacc 659

<210> 3

<211> 171

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 3

gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 60

agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 120

ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt c 171

<210> 4

<211> 220

<212> DNA

<213> Human cytomegalovirus (Human cytomegavirus)

<400> 4

tgctgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat 60

ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 120

actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac 180

ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 220

<210> 5

<211> 923

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 5

gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 60

ccttgaatta cttccacctg gctccagtac gtgattcttg atcccgagct ggagccaggg 120

gcgggccttg cgctttagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc 180

gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata 240

agtctctagc catttaaaat ttttgatgac gtgctgcgac gctttttttc tggcaagata 300

gtcttgtaaa tgcgggccag gatctgcaca ctggtatttc ggtttttggg cccgcggccg 360

gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc 420

caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg 480

cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt 540

gagcggaaag atggccgctt cccggccctg ctccaggggg ctcaaaatgg aggacgcggc 600

gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag 660

ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct 720

ggagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc 780

cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct 840

tggaatttgg cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc 900

aaagtttttt tcttccattt cag 923

<210> 6

<211> 27

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 6

acacccaagc tgtctagagc cgccacc 27

<210> 7

<211> 300

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 7

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 60

tcatcatttt cccattgtaa aatgccatat ttttttcata ggtcacttac atatatcaat 120

gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc cacacatctc 180

atgctttgct ctaaatcttg atatttagtg gaacattctt tcccattttg ttctacaaga 240

atatttttgt tattgtcttt gggctttcta tatacatttt gaaatgaggt tgacaagtta 300

<210> 8

<211> 589

<212> DNA

<213> Woodchuck hepatitis virus (Woodchuck hepatitis virus)

<400> 8

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcctctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589

<210> 9

<211> 251

<212> DNA

<213> cattle (Bos taurus)

<400> 9

ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 60

tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 120

ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aatacaatag 180

caggcatgct ggggatgcgg tgggctctat gggtacccag gtgctgaaga attgacccgg 240

ttcctcctgg g 251

<210> 10

<211> 127

<212> DNA

<213> Adeno-associated virus 2 (Adeno-associated virus 2)

<400> 10

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttcc 127

<210> 11

<211> 318

<212> DNA

<213> Human adenovirus 2 (Human adenoviruses 2)

<400> 11

ctcactctct tccgcatcgc tgtctgcgag ggccagctgt tgggctcgcg gttgaggaca 60

aactcttcgc ggtctttcca gtactcttgg atcggaaacc cgtcggcctc cgaacggtac 120

tccgccaccg agggacctga gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa 180

ggcgtctaac cagtcacagt cgcaaggtag gctgagcacc gtggcgggcg gcagcgggtg 240

gcggtcgggg ttgtttctgg cggaggtgct gctgatgatg taattaaagt aggcggtctt 300

gagacggcgg atggtcga 318

<210> 12

<211> 102

<212> DNA

<213> Human adenovirus 2 (Human adenoviruses 2)

<400> 12

ccagctgttg gggtgagtac tccctctcaa aagcgggcat tacttctgcg ctaagattgt 60

cagtttccaa aaacgaggag gatttgatat tcacctggcc cg 102

<210> 13

<211> 810

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 13

ctgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60

gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120

tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180

tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240

tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300

tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360

gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420

atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480

aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540

atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600

ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660

tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720

gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780

tatacatttt gaaatgaggt tgacaagtta 810

<210> 14

<211> 202

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 14

ctgcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc 60

actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag 120

gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcccaagttg 180

ggaagaaacc tgtagggcct gc 202

<210> 15

<211> 145

<212> DNA

<213> Adeno-associated virus 2 (Adeno-associated virus 2)

<400> 15

gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc 60

tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 120

ggcctcagtg agcgagcgag cgcgc 145

<210> 16

<211> 155

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 16

ggcatgcttc tatattattt tctaaaagat ttaaagtttt gccttctcca tttagactta 60

taattcactg gaattttttt gtgtgtatgg tatgacatat gggttccctt ttatttttta 120

catataaata tatttccctg tttttctaaa aaaga 155

<210> 17

<211> 726

<212> DNA

<213> Human hepatitis B Virus (Human hepatitis B virus)

<400> 17

ataacaggcc tattgattgg aaagtttgtc aacgaattgt gggtcttttg gggtttgctg 60

ccccttttac gcaatgtgga tatcctgctt taatgccttt atatgcatgt atacaagcaa 120

aacaggcttt tactttctcg ccaacttaca aggcctttct cagtaaacag tatatgaccc 180

tttaccccgt tgctcggcaa cggcctggtc tgtgccaagt gtttgctgac gcaaccccca 240

ctggttgggg cttggccata ggccatcagc gcatgcgtgg aacctttgtg tctcctctgc 300

cgatccatac tgcggaactc ctagccgctt gttttgctcg cagcaggtct ggagcaaacc 360

tcatcgggac cgacaattct gtcgtactct cccgcaagta tacatcgttt ccatggctgc 420

taggctgtgc tgccaactgg atcctgcgcg ggacgtcctt tgtttacgtc ccgtcggcgc 480

tgaatcccgc ggacgacccc tcccggggcc gcttggggct ctaccgcccg cttctccgtc 540

tgccgtaccg tccgaccacg gggcgcacct ctctttacgc ggactccccg tctgtgcctt 600

ctcatctgcc ggaccgtgtg cacttcgctt cacctctgca cgtcgcatgg aggccaccgt 660

gaacgcccac cggaacctgc ccaaggtctt gcataagagg actcttggac tttcagcaat 720

gtcatc 726

<210> 18

<211> 170

<212> DNA

<213> Human cytomegalovirus (Human cytomegavirus)

<400> 18

gtaagtctgt tgacatgtat gtgatgtata ctaacctgca tgggacgtgg atttacttgt 60

gtatgtcaga tagagtaaag attaactctt gcatgtgagc ggggcatcga gatagcgata 120

aatgagtcag gaggacggat acttatatgt gttgttatcc tcctctacag 170

<210> 19

<211> 50

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 19

agcttgcttg ttctttttgc agaagctcag aataaacgct caactttggc 50

<210> 20

<211> 387

<212> DNA

<213> Rabbit (Oryctolagus cuniculus)

<400> 20

tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc 60

actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta 120

gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg 180

tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac 240

ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa 300

attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat 360

agctgtccct cttctcttat ggagatc 387

<210> 21

<211> 83

<212> DNA

<213> Human cytomegalovirus (Human cytomegavirus)

<400> 21

ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 60

ctcgtttagt gaaccgtcag atc 83

<210> 22

<211> 133

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> synthetic construct

<400> 22

gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60

cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120

tttctctcca cag 133

<210> 23

<211> 197

<212> DNA

<213> Human cytomegalovirus (Human cytomegavirus)

<400> 23

ggggctcggg tgcagcggcc agcgggcgcc tggcggcgag gattacccgg ggaagtggtt 60

gtctcctggc tggagccgcg agacgggcgc tcagggcgcg gggccggcgg cggcgaacga 120

gaggacggac tctggcggcc gggtctttgg ccgcggggag cgcgggcacc gggcgagcag 180

gccgcgtcgc gctcacc 197

<210> 24

<211> 2064

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 24

atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60

acaggatcta gttcaggttc aaaattaaaa gatcctgaac tgagtttaaa aggcacccag 120

cacatcatgc aagcaggcca gacactgcat ctccaatgca ggggggaagc agcccataaa 180

tggtctttgc ctgaaatggt gagtaaggaa agcgaaaggc tgagcataac taaatctgcc 240

tgtggaagaa atggcaaaca attctgcagt actttaacct tgaacacagc tcaagcaaac 300

cacactggct tctacagctg caaatatcta gctgtaccta cttcaaagaa gaaggaaaca 360

gaatctgcaa tctatatatt tattagtgat acaggtagac ctttcgtaga gatgtacagt 420

gaaatccccg aaattataca catgactgaa ggaagggagc tcgtcattcc ctgccgggtt 480

acgtcaccta acatcactgt tactttaaaa aagtttccac ttgacacttt gatccctgat 540

ggaaaacgca taatctggga cagtagaaag ggcttcatca tatcaaatgc aacgtacaaa 600

gaaatagggc ttctgacctg tgaagcaaca gtcaatgggc atttgtataa gacaaactat 660

ctcacacatc gacaaaccaa tacaatcata gatgtccaaa taagcacacc acgcccagtc 720

aaattactta gaggccatac tcttgtcctc aattgtactg ctaccactcc cttgaacacg 780

agagttcaaa tgacctggag ttaccctgat gaaaaaaata agagagcttc cgtaaggcga 840

cgaattgacc aaagcaattc ccatgccaac atattctaca gtgttcttac tattgacaaa 900

atgcagaaca aagacaaagg actttatact tgtcgtgtaa ggagtggacc atcattcaaa 960

tctgttaaca cctcagtgca tatatatgat aaagcattca tcactgtgaa acatcgaaaa 1020

cagcaggtgc ttgaaaccgt agctggcaag cggtcttacc ggctctctat gaaagtgaag 1080

gcatttccct cgccggaagt tgtatggtta aaagatgggt tacctgcgac tgagaaatct 1140

gctcgctatt tgactcgtgg ctactcgtta attatcaagg acgtaactga agaggatgca 1200

gggaattata caatcttgct gagcataaaa cagtcaaatg tgtttaaaaa cctcactgcc 1260

actctaattg tcaatgtgaa accccagatt tacgaaaagg ccgtgtcatc gtttccagac 1320

ccggctctct acccactggg cagcagacaa atcctgactt gtaccgcata tggtatccct 1380

caacctacaa tcaagtggtt ctggcacccc tgtaaccata atcattccga agcaaggtgt 1440

gacttttgtt ccaataatga agagtccttt atcctggatg ctgacagcaa catgggaaac 1500

agaattgaga gcatcactca gcgcatggca ataatagaag gaaagaataa gatggctagc 1560

accttggttg tggctgactc tagaatttct ggaatctaca tttgcatagc ttccaataaa 1620

gttgggactg tgggaagaaa cataagcttt tatatcacag atgtgccaaa tgggtttcat 1680

gttaacttgg aaaaaatgcc gacggaagga gaggacctga aactgtcttg cacagttaac 1740

aagttcttat acagagacgt tacttggatt ttactgcgga cagttaataa cagaacaatg 1800

cactacagta ttagcaagca aaaaatggcc atcactaagg agcactccat cactcttaat 1860

cttaccatca tgaatgtttc cctgcaagat tcaggcacct atgcctgcag agccaggaat 1920

gtatacacag gggaagaaat cctccagaag aaagaaatta caatcagagg tgagcactgc 1980

aacaaaaagg ctgttttctc tcggatctcc aaatttaaaa gcacaaggaa tgattgtacc 2040

acacaaagta atgtaaaaca ttaa 2064

<210> 25

<211> 20

<212> DNA

<213> Human cytomegalovirus (Human cytomegavirus)

<400> 25

aggactcatt aaaaagtaac 20

<210> 26

<211> 222

<212> DNA

<213> Simian Virus 40 (Simian Virus 40)

<400> 26

cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 60

aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 120

ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt 180

gggaggtttt ttaaagcaag taaaacctct acaaatgtgg ta 222

<210> 27

<211> 1075

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 1 complete 5'

<400> 27

ctctggagac gcaacctttg gagctaagcc agcaatggta gagggaagat tctgcacgtc 60

ccttccaggc ggcctccccg tcaccacccc ccccaacccg ccccgaccgg agctgagagt 120

aattcataca aaaggactcg cccctgcctt ggggaatccc agggaccgtc gttaaactcc 180

cactaacgta gaacccagag atcgctgcgt tcccgccccc tcacccgccc gctctcgtca 240

tcactgaggt ggagaatagc atgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac 300

atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag 360

aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga 420

gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg 480

gtttgccgcc agaacacagc gtctcagggg agatctcgtt tagtgaaccg tcagatcctc 540

actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 600

tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acggtactcc 660

gccaccgagg gacctgagcg agtccgcatc gaccggatcg gaaaacctct cgagaaaggc 720

gtctaaccag tcacagtcgc aaggtaggct gagcaccgtg gcgggcggca gcgggtggcg 780

gtcggggttg tttctggcgg aggtgctgct gatgatgtaa ttaaagtagg cggtcttgag 840

acggcggatg gtcgaggtga ggtgtggcag gcttgagatc cagctgttgg ggtgagtact 900

ccctctcaaa agcgggcatt acttctgcgc taagattgtc agtttccaaa aacgaggagg 960

atttgatatt cacctggccc gatctggcca tacacttgag tgacaatgac atccactttg 1020

cctttctctc cacaggtgtc cactcccagg tccaagttta aacgccgcca ccatg 1075

<210> 28

<211> 1768

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 1 complete 3'

<400> 28

ctgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60

gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120

tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180

tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240

tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300

tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360

gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420

atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480

aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540

atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600

ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660

tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720

gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780

tatacatttt gaaatgaggt tgacaagtta cctaggaaaa ctgtcttcat aacaggccta 840

ttgattggaa agtttgtcaa cgaattgtgg gtcttttggg gtttgctgcc ccttttacgc 900

aatgtggata tcctgcttta atgcctttat atgcatgtat acaagcaaaa caggctttta 960

ctttctcgcc aacttacaag gcctttctca gtaaacagta tatgaccctt taccccgttg 1020

ctcggcaacg gcctggtctg tgccaagtgt ttgctgacgc aacccccact ggttggggct 1080

tggccatagg ccatcagcgc atgcgtggaa cctttgtgtc tcctctgccg atccatactg 1140

cggaactcct agccgcttgt tttgctcgca gcaggtctgg agcaaacctc atcgggaccg 1200

acaattctgt cgtactctcc cgcaagtata catcgtttcc atggctgcta ggctgtgctg 1260

ccaactggat cctgcgcggg acgtcctttg tttacgtccc gtcggcgctg aatcccgcgg 1320

acgacccctc ccggggccgc ttggggctct accgcccgct tctccgtctg ccgtaccgtc 1380

cgaccacggg gcgcacctct ctttacgcgg actccccgtc tgtgccttct catctgccgg 1440

accgtgtgca cttcgcttca cctctgcacg tcgcatggag gccaccgtga acgcccaccg 1500

gaacctgccc aaggtcttgc ataagaggac tcttggactt tcagcaatgt catcctgccc 1560

gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 1620

gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 1680

ttctataata ttatggggtg gaggggggtg gtatggagca aggggcccaa gttgggaaga 1740

aacctgtagg gcctgcgaag acagtcag 1768

<210> 29

<211> 1193

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 2 complete 5'

<400> 29

ctctggagac ggcacatcgc ccacagtccc cgagaagttg ggaggggtcg gcaattgaac 60

cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg 120

cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct 180

ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc 240

tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctccagta 300

cgtgattctt gatcccgagc tggagccagg ggcgggcctt gcgctttagg agccccttcg 360

cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg 420

caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga 480

cgtgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca ggatctgcac 540

actggtattt cggtttttgg gcccgcggcc ggcgacgggg cccgtgcgtc ccagcgcaca 600

tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa 660

gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg 720

gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct 780

gctccagggg gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc 840

acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac 900

cgggcgccgt ccaggcacct cgattagttc tggagctttt ggagtacgtc gtctttaggt 960

tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt 1020

aggccagctt ggcacttgat gtaattctcc ttggaatttg gcctttttga gtttggatct 1080

tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg 1140

tgaacacgtc tcaggggaga tctacaccca agctgtctag agccgccacc atg 1193

<210> 30

<211> 984

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 2 complete 3'

<400> 30

tgaggcatgc ttctatatta ttttctaaaa gatttaaagt tttgccttct ccatttagac 60

ttataattca ctggaatttt tttgtgtgta tggtatgaca tatgggttcc cttttatttt 120

ttacatataa atatatttcc ctgtttttct aaaaaagacc taggaaaact gtcttcataa 180

tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 240

ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 300

ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 360

gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 420

ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 480

tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 540

gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 600

ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 660

tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcctcttcg 720

ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc ctgcccgggt 780

ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc 840

ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct 900

ataatattat ggggtggagg ggggtggtat ggagcaaggg gcccaagttg ggaagaaacc 960

tgtagggcct gcgaagacag tcag 984

<210> 31

<211> 1481

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 3 complete 5'

<400> 31

ctctggagac gcaacctttg gagctaagcc agcaatggta gagggaagat tctgcacgtc 60

ccttccaggc ggcctccccg tcaccacccc ccccaacccg ccccgaccgg agctgagagt 120

aattcataca aaaggactcg cccctgcctt ggggaatccc agggaccgtc gttaaactcc 180

cactaacgta gaacccagag atcgctgcgt tcccgccccc tcacccgccc gctctcgtca 240

tcactgaggt ggagaatagc atgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac 300

atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag 360

aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga 420

gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg 480

gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac 540

gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctccagtacg tgattcttga 600

tcccgagctg gagccagggg cgggccttgc gctttaggag ccccttcgcc tcgtgcttga 660

gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc 720

tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacg tgctgcgacg 780

ctttttttct ggcaagatag tcttgtaaat gcgggccagg atctgcacac tggtatttcg 840

gtttttgggc ccgcggccgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg 900

cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct 960

gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc 1020

cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tccagggggc 1080

tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa 1140

agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc 1200

aggcacctcg attagttctg gagcttttgg agtacgtcgt ctttaggttg gggggagggg 1260

ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg 1320

cacttgatgt aattctcctt ggaatttggc ctttttgagt ttggatcttg gttcattctc 1380

aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg aacacgtctc 1440

aggggagatc tacacccaag ctgtctagag ccgccaccat g 1481

<210> 32

<211> 581

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 3 complete 3'

<400> 32

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 60

tcatcatttt cccattgtaa aatgccatat ttttttcata ggtcacttac atatatcaat 120

gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc cacacatctc 180

atgctttgct ctaaatcttg atatttagtg gaacattctt tcccattttg ttctacaaga 240

atatttttgt tattgtcttt gggctttcta tatacatttt gaaatgaggt tgacaagtta 300

cctaggaaaa ctgtcttctt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 360

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 420

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 480

ggattgggaa tacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtacccaggt 540

gctgaagaat tgacccggtt cctcctgggg aagacagtca g 581

<210> 33

<211> 1511

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 4 complete 5'

<400> 33

ctctggagac gcaacctttg gagctaagcc agcaatggta gagggaagat tctgcacgtc 60

ccttccaggc ggcctccccg tcaccacccc ccccaacccg ccccgaccgg agctgagagt 120

aattcataca aaaggactcg cccctgcctt ggggaatccc agggaccgtc gttaaactcc 180

cactaacgta gaacccagag atcgctgcgt tcccgccccc tcacccgccc gctctcgtca 240

tcactgaggt ggagaatagc atgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac 300

atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag 360

aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga 420

gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg 480

gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac 540

gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctccagtacg tgattcttga 600

tcccgagctg gagccagggg cgggccttgc gctttaggag ccccttcgcc tcgtgcttga 660

gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc 720

tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacg tgctgcgacg 780

ctttttttct ggcaagatag tcttgtaaat gcgggccagg atctgcacac tggtatttcg 840

gtttttgggc ccgcggccgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg 900

cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct 960

gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc 1020

cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tccagggggc 1080

tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa 1140

agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc 1200

aggcacctcg attagttctg gagcttttgg agtacgtcgt ctttaggttg gggggagggg 1260

ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg 1320

cacttgatgt aattctcctt ggaatttggc ctttttgagt ttggatcttg gttcattctc 1380

aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg aacacgtctc 1440

aggggagatc tagcttgctt gttctttttg cagaagctca gaataaacgc tcaactttgg 1500

ccgccaccat g 1511

<210> 34

<211> 717

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 4 complete 3'

<400> 34

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 60

tcatcatttt cccattgtaa aatgccatat ttttttcata ggtcacttac atatatcaat 120

gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc cacacatctc 180

atgctttgct ctaaatcttg atatttagtg gaacattctt tcccattttg ttctacaaga 240

atatttttgt tattgtcttt gggctttcta tatacatttt gaaatgaggt tgacaagtta 300

cctaggaaaa ctgtcttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg 360

gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca 420

gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa 480

aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt 540

ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt 600

ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc 660

ctgactactc ccagtcatag ctgtccctct tctcttatgg agatcgaaga cagtcag 717

<210> 35

<211> 928

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 5 complete 5'

<400> 35

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accaatgacg tcgaggagaa gttccccaac tttcccgcct ctcagccttt gaaagaaaga 360

aaggggaggg ggcaggccgc gtgcagccgc gagcggtgct gggctccggc tccaattccc 420

catctcagtc gttcccaaag tcctcctgtt tcatccaagc gtgtaagggt ccccgtcctt 480

gactccctag tgtcctgctg cccacagtcc agtcctggga accagcaccg atcacctccc 540

atcgggccaa tctcagtccc ttccccccta cgtcggggcc cacacgctcg gtgcgtgccc 600

agttgaacca ggcggctgcg gaaaaaaaaa agcggggaga aagtagggcc cggctactag 660

cggttttacg ggcgcacgta gctcaggcct caagaccttg ggctgggact ggctgagcct 720

ggcgggaggc ggggtccgag tcaccgcctg ccgccgcgcc cccggtttct ataaattgag 780

cccgcagcct cccgcttcgc tctctgctcc tcctgttcga cagtcagccg catcttcttt 840

tgcgtcgcca gcgtctcagg ggagatctag cttgcttgtt ctttttgcag aagctcagaa 900

taaacgctca actttggccg ccaccatg 928

<210> 36

<211> 1268

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 5 complete 3'

<400> 36

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 60

tcatcatttt cccattgtaa aatgccatat ttttttcata ggtcacttac atatatcaat 120

gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc cacacatctc 180

atgctttgct ctaaatcttg atatttagtg gaacattctt tcccattttg ttctacaaga 240

atatttttgt tattgtcttt gggctttcta tatacatttt gaaatgaggt tgacaagtta 300

cctaggaaaa ctgtcttcat aacaggccta ttgattggaa agtttgtcaa cgaattgtgg 360

gtcttttggg gtttgctgcc ccttttacgc aatgtggata tcctgcttta atgcctttat 420

atgcatgtat acaagcaaaa caggctttta ctttctcgcc aacttacaag gcctttctca 480

gtaaacagta tatgaccctt taccccgttg ctcggcaacg gcctggtctg tgccaagtgt 540

ttgctgacgc aacccccact ggttggggct tggccatagg ccatcagcgc atgcgtggaa 600

cctttgtgtc tcctctgccg atccatactg cggaactcct agccgcttgt tttgctcgca 660

gcaggtctgg agcaaacctc atcgggaccg acaattctgt cgtactctcc cgcaagtata 720

catcgtttcc atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg 780

tttacgtccc gtcggcgctg aatcccgcgg acgacccctc ccggggccgc ttggggctct 840

accgcccgct tctccgtctg ccgtaccgtc cgaccacggg gcgcacctct ctttacgcgg 900

actccccgtc tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg 960

tcgcatggag gccaccgtga acgcccaccg gaacctgccc aaggtcttgc ataagaggac 1020

tcttggactt tcagcaatgt catcctgccc gggtggcatc cctgtgaccc ctccccagtg 1080

cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta 1140

agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg 1200

gtatggagca aggggcccaa gttgggaaga aacctgtagg gcctgcgaag acagtcagac 1260

tgtcatta 1268

<210> 37

<211> 1427

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 6 complete 5'

<400> 37

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accaatgacg tcgaggagaa gttccccaac tttcccgcct ctcagccttt gaaagaaaga 360

aaggggaggg ggcaggccgc gtgcagccgc gagcggtgct gggctccggc tccaattccc 420

catctcagtc gttcccaaag tcctcctgtt tcatccaagc gtgtaagggt ccccgtcctt 480

gactccctag tgtcctgctg cccacagtcc agtcctggga accagcaccg atcacctccc 540

atcgggccaa tctcagtccc ttccccccta cgtcggggcc cacacgctcg gtgcgtgccc 600

agttgaacca ggcggctgcg gaaaaaaaaa agcggggaga aagtagggcc cggctactag 660

cggttttacg ggcgcacgta gctcaggcct caagaccttg ggctgggact ggctgagcct 720

ggcgggaggc ggggtccgag tcaccgcctg ccgccgcgcc cccggtttct ataaattgag 780

cccgcagcct cccgcttcgc tctctgctcc tcctgttcga cagtcagccg catcttcttt 840

tgcgtcgcca gcgtctcagg ggagatctcg tttagtgaac cgtcagatcc tcactctctt 900

ccgcatcgct gtctgcgagg gccagctgtt gggctcgcgg ttgaggacaa actcttcgcg 960

gtctttccag tactcttgga tcggaaaccc gtcggcctcc gaacggtact ccgccaccga 1020

gggacctgag cgagtccgca tcgaccggat cggaaaacct ctcgagaaag gcgtctaacc 1080

agtcacagtc gcaaggtagg ctgagcaccg tggcgggcgg cagcgggtgg cggtcggggt 1140

tgtttctggc ggaggtgctg ctgatgatgt aattaaagta ggcggtcttg agacggcgga 1200

tggtcgaggt gaggtgtggc aggcttgaga tccagctgtt ggggtgagta ctccctctca 1260

aaagcgggca ttacttctgc gctaagattg tcagtttcca aaaacgagga ggatttgata 1320

ttcacctggc ccgatctggc catacacttg agtgacaatg acatccactt tgcctttctc 1380

tccacaggtg tccactccca ggtccaagtt taaacgccgc caccatg 1427

<210> 38

<211> 1169

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 6 complete 3'

<400> 38

tgaggcatgc ttctatatta ttttctaaaa gatttaaagt tttgccttct ccatttagac 60

ttataattca ctggaatttt tttgtgtgta tggtatgaca tatgggttcc cttttatttt 120

ttacatataa atatatttcc ctgtttttct aaaaaagacc taggaaaact gtcttcataa 180

tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 240

ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 300

ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 360

gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 420

ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 480

tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 540

gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 600

ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 660

tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcctcttcg 720

ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc tggctaataa 780

aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg 840

acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca 900

acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat 960

atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag 1020

atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta 1080

catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct 1140

cttctcttat ggagatcgaa gacagtcag 1169

<210> 39

<211> 1755

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 7 complete 5'

<400> 39

ctctggagac gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 60

ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 120

cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 180

tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 240

tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 300

atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc 360

ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg 420

gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 480

gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 540

ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 600

tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 660

ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg 720

taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg aaagccttga 780

ggggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg 840

tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc 900

gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg 960

ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg 1020

gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc 1080

cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg 1140

ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc 1200

ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc 1260

gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac 1320

ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta 1380

gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc 1440

gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg 1500

acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1560

gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1620

acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcggcttg atcgaagccg 1680

tctcagggga gatctagctt gcttgttctt tttgcagaag ctcagaataa acgctcaact 1740

ttggccgcca ccatg 1755

<210> 40

<211> 1165

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 7 complete 3'

<400> 40

tgaggcatgc ttctatatta ttttctaaaa gatttaaagt tttgccttct ccatttagac 60

ttataattca ctggaatttt tttgtgtgta tggtatgaca tatgggttcc cttttatttt 120

ttacatataa atatatttcc ctgtttttct aaaaaagacc taggaaaact gtcttcataa 180

caggcctatt gattggaaag tttgtcaacg aattgtgggt cttttggggt ttgctgcccc 240

ttttacgcaa tgtggatatc ctgctttaat gcctttatat gcatgtatac aagcaaaaca 300

ggcttttact ttctcgccaa cttacaaggc ctttctcagt aaacagtata tgacccttta 360

ccccgttgct cggcaacggc ctggtctgtg ccaagtgttt gctgacgcaa cccccactgg 420

ttggggcttg gccataggcc atcagcgcat gcgtggaacc tttgtgtctc ctctgccgat 480

ccatactgcg gaactcctag ccgcttgttt tgctcgcagc aggtctggag caaacctcat 540

cgggaccgac aattctgtcg tactctcccg caagtataca tcgtttccat ggctgctagg 600

ctgtgctgcc aactggatcc tgcgcgggac gtcctttgtt tacgtcccgt cggcgctgaa 660

tcccgcggac gacccctccc ggggccgctt ggggctctac cgcccgcttc tccgtctgcc 720

gtaccgtccg accacggggc gcacctctct ttacgcggac tccccgtctg tgccttctca 780

tctgccggac cgtgtgcact tcgcttcacc tctgcacgtc gcatggaggc caccgtgaac 840

gcccaccgga acctgcccaa ggtcttgcat aagaggactc ttggactttc agcaatgtca 900

tcttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 960

actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 1020

cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaatacaat 1080

agcaggcatg ctggggatgc ggtgggctct atgggtaccc aggtgctgaa gaattgaccc 1140

ggttcctcct ggggaagaca gtcag 1165

<210> 41

<211> 1159

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 8 complete 5'

<400> 41

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 360

cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 420

ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga 480

ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg 540

cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cggcgtctca ggggagatct 600

cgtttagtga accgtcagat cctcactctc ttccgcatcg ctgtctgcga gggccagctg 660

ttgggctcgc ggttgaggac aaactcttcg cggtctttcc agtactcttg gatcggaaac 720

ccgtcggcct ccgaacggta ctccgccacc gagggacctg agcgagtccg catcgaccgg 780

atcggaaaac ctctcgagaa aggcgtctaa ccagtcacag tcgcaaggta ggctgagcac 840

cgtggcgggc ggcagcgggt ggcggtcggg gttgtttctg gcggaggtgc tgctgatgat 900

gtaattaaag taggcggtct tgagacggcg gatggtcgag gtgaggtgtg gcaggcttga 960

gatccagctg ttggggtgag tactccctct caaaagcggg cattacttct gcgctaagat 1020

tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgatctg gccatacact 1080

tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaag 1140

tttaaacgcc gccaccatg 1159

<210> 42

<211> 572

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 8 complete 3'

<400> 42

ggcatgcttc tatattattt tctaaaagat ttaaagtttt gccttctcca tttagactta 60

taattcactg gaattttttt gtgtgtatgg tatgacatat gggttccctt ttatttttta 120

catataaata tatttccctg tttttctaaa aaagacctag gaaaactgtc ttctggctaa 180

taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga 240

aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg 300

gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag 360

tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt 420

tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc 480

ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc 540

cctcttctct tatggagatc gaagacagtc ag 572

<210> 43

<211> 1519

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 9 complete 5'

<400> 43

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accagcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt 360

gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 420

tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 480

cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc 540

ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct ccagtacgtg 600

attcttgatc ccgagctgga gccaggggcg ggccttgcgc tttaggagcc ccttcgcctc 660

gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc 720

ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacgtg 780

ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaggat ctgcacactg 840

gtatttcggt ttttgggccc gcggccggcg acggggcccg tgcgtcccag cgcacatgtt 900

cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg 960

gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa 1020

ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctc 1080

cagggggctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac 1140

aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg 1200

cgccgtccag gcacctcgat tagttctgga gcttttggag tacgtcgtct ttaggttggg 1260

gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc 1320

cagcttggca cttgatgtaa ttctccttgg aatttggcct ttttgagttt ggatcttggt 1380

tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcctgaa 1440

cacgtctcag gggagatcta gcttgcttgt tctttttgca gaagctcaga ataaacgctc 1500

aactttggcc gccaccatg 1519

<210> 44

<211> 387

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 9 complete 3'

<400> 44

ggcatgcttc tatattattt tctaaaagat ttaaagtttt gccttctcca tttagactta 60

taattcactg gaattttttt gtgtgtatgg tatgacatat gggttccctt ttatttttta 120

catataaata tatttccctg tttttctaaa aaagacctag gaaaactgtc ttcctgcccg 180

ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 240

tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 300

tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcccaag ttgggaagaa 360

acctgtaggg cctgcgaaga cagtcag 387

<210> 45

<211> 1489

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 10 complete 5'

<400> 45

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accagcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt 360

gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 420

tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 480

cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc 540

ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct ccagtacgtg 600

attcttgatc ccgagctgga gccaggggcg ggccttgcgc tttaggagcc ccttcgcctc 660

gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc 720

ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacgtg 780

ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaggat ctgcacactg 840

gtatttcggt ttttgggccc gcggccggcg acggggcccg tgcgtcccag cgcacatgtt 900

cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg 960

gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa 1020

ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctc 1080

cagggggctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac 1140

aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg 1200

cgccgtccag gcacctcgat tagttctgga gcttttggag tacgtcgtct ttaggttggg 1260

gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc 1320

cagcttggca cttgatgtaa ttctccttgg aatttggcct ttttgagttt ggatcttggt 1380

tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcctgaa 1440

cacgtctcag gggagatcta cacccaagct gtctagagcc gccaccatg 1489

<210> 46

<211> 1180

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 10 complete 3'

<400> 46

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 60

tcatcatttt cccattgtaa aatgccatat ttttttcata ggtcacttac atatatcaat 120

gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc cacacatctc 180

atgctttgct ctaaatcttg atatttagtg gaacattctt tcccattttg ttctacaaga 240

atatttttgt tattgtcttt gggctttcta tatacatttt gaaatgaggt tgacaagtta 300

cctaggaaaa ctgtcttcat ataatcaacc tctggattac aaaatttgtg aaagattgac 360

tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt 420

gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt 480

gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt 540

gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg 600

gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg 660

ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc 720

atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt 780

ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc 840

tctgcggcct cttccgcctc ttcgccttcg ccctcagacg agtcggatct ccctttgggc 900

cgcctccccg catcatcttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 960

accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 1020

tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 1080

gattgggaat acaatagcag gcatgctggg gatgcggtgg gctctatggg tacccaggtg 1140

ctgaagaatt gacccggttc ctcctgggga agacagtcag 1180

<210> 47

<211> 1172

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 11 complete 5'

<400> 47

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatgctga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 360

ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 420

cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt 480

gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540

ggccatccac gctgttttga cctccatagt ggacaccggg accgatccag cctccgcgtc 600

tcaggggaga tctcgtttag tgaaccgtca gatcctcact ctcttccgca tcgctgtctg 660

cgagggccag ctgttgggct cgcggttgag gacaaactct tcgcggtctt tccagtactc 720

ttggatcgga aacccgtcgg cctccgaacg gtactccgcc accgagggac ctgagcgagt 780

ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc taaccagtca cagtcgcaag 840

gtaggctgag caccgtggcg ggcggcagcg ggtggcggtc ggggttgttt ctggcggagg 900

tgctgctgat gatgtaatta aagtaggcgg tcttgagacg gcggatggtc gaggtgaggt 960

gtggcaggct tgagatccag ctgttggggt gagtactccc tctcaaaagc gggcattact 1020

tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac ctggcccgat 1080

ctggccatac acttgagtga caatgacatc cactttgcct ttctctccac aggtgtccac 1140

tcccaggtcc aagtttaaac gccgccacca tg 1172

<210> 48

<211> 1043

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 11 complete 3'

<400> 48

actgttctca tcacatcata tcaaggttat ataccatcaa tattgccaca gatgttactt 60

agccttttaa tatttctcta atttagtgta tatgcaatga tagttctctg atttctgaga 120

ttgagtttct catgtgtaat gattatttag agtttctctt tcatctgttc aaatttttgt 180

ctagttttat tttttactga tttgtaagac ttctttttat aatctgcata ttacaattct 240

ctttactggg gtgttgcaaa tattttctgt cattctatgg cctgactttt cttaatggtt 300

ttttaatttt aaaaataagt cttaatattc atgcaatcta attaacaatc ttttctttgt 360

ggttaggact ttgagtcata agaaattttt ctctacactg aagtcatgat ggcatgcttc 420

tatattattt tctaaaagat ttaaagtttt gccttctcca tttagactta taattcactg 480

gaattttttt gtgtgtatgg tatgacatat gggttccctt ttatttttta catataaata 540

tatttccctg tttttctaaa aaagaaaaag atcatcattt tcccattgta aaatgccata 600

tttttttcat aggtcactta catatatcaa tgggtctgtt tctgagctct actctatttt 660

atcagcctca ctgtctatcc ccacacatct catgctttgc tctaaatctt gatatttagt 720

ggaacattct ttcccatttt gttctacaag aatatttttg ttattgtctt tgggctttct 780

atatacattt tgaaatgagg ttgacaagtt acctaggaaa actgtcttcc tgcccgggtg 840

gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc 900

caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta 960

taatattatg gggtggaggg gggtggtatg gagcaagggg cccaagttgg gaagaaacct 1020

gtagggcctg cgaagacagt cag 1043

<210> 49

<211> 1172

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 12 complete 5'

<400> 49

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatgctga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 360

ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 420

cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt 480

gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540

ggccatccac gctgttttga cctccatagt ggacaccggg accgatccag cctccgcgtc 600

tcaggggaga tctcgtttag tgaaccgtca gatcctcact ctcttccgca tcgctgtctg 660

cgagggccag ctgttgggct cgcggttgag gacaaactct tcgcggtctt tccagtactc 720

ttggatcgga aacccgtcgg cctccgaacg gtactccgcc accgagggac ctgagcgagt 780

ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc taaccagtca cagtcgcaag 840

gtaggctgag caccgtggcg ggcggcagcg ggtggcggtc ggggttgttt ctggcggagg 900

tgctgctgat gatgtaatta aagtaggcgg tcttgagacg gcggatggtc gaggtgaggt 960

gtggcaggct tgagatccag ctgttggggt gagtactccc tctcaaaagc gggcattact 1020

tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac ctggcccgat 1080

ctggccatac acttgagtga caatgacatc cactttgcct ttctctccac aggtgtccac 1140

tcccaggtcc aagtttaaac gccgccacca tg 1172

<210> 50

<211> 1165

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 12 complete 3'

<400> 50

tgaggcatgc ttctatatta ttttctaaaa gatttaaagt tttgccttct ccatttagac 60

ttataattca ctggaatttt tttgtgtgta tggtatgaca tatgggttcc cttttatttt 120

ttacatataa atatatttcc ctgtttttct aaaaaagacc taggaaaact gtcttcataa 180

caggcctatt gattggaaag tttgtcaacg aattgtgggt cttttggggt ttgctgcccc 240

ttttacgcaa tgtggatatc ctgctttaat gcctttatat gcatgtatac aagcaaaaca 300

ggcttttact ttctcgccaa cttacaaggc ctttctcagt aaacagtata tgacccttta 360

ccccgttgct cggcaacggc ctggtctgtg ccaagtgttt gctgacgcaa cccccactgg 420

ttggggcttg gccataggcc atcagcgcat gcgtggaacc tttgtgtctc ctctgccgat 480

ccatactgcg gaactcctag ccgcttgttt tgctcgcagc aggtctggag caaacctcat 540

cgggaccgac aattctgtcg tactctcccg caagtataca tcgtttccat ggctgctagg 600

ctgtgctgcc aactggatcc tgcgcgggac gtcctttgtt tacgtcccgt cggcgctgaa 660

tcccgcggac gacccctccc ggggccgctt ggggctctac cgcccgcttc tccgtctgcc 720

gtaccgtccg accacggggc gcacctctct ttacgcggac tccccgtctg tgccttctca 780

tctgccggac cgtgtgcact tcgcttcacc tctgcacgtc gcatggaggc caccgtgaac 840

gcccaccgga acctgcccaa ggtcttgcat aagaggactc ttggactttc agcaatgtca 900

tcttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 960

actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 1020

cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaatacaat 1080

agcaggcatg ctggggatgc ggtgggctct atgggtaccc aggtgctgaa gaattgaccc 1140

ggttcctcct ggggaagaca gtcag 1165

<210> 51

<211> 820

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 13 complete 5'

<400> 51

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 360

cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 420

ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga 480

ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg 540

cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cggcgtctca ggggagatct 600

ggtgaggtgt ggcaggcttg agatccagct gttggggtga gtactccctc tcaaaagcgg 660

gcattacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcacct 720

ggcccgatct ggccatacac ttgagtgaca atgacatcca ctttgccttt ctctccacag 780

gtgtccactc ccaggtccaa gtttaaacgc cgccaccatg 820

<210> 52

<211> 1443

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 13 complete 3'

<400> 52

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 60

tcatcatttt cccattgtaa aatgccatat ttttttcata ggtcacttac atatatcaat 120

gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc cacacatctc 180

atgctttgct ctaaatcttg atatttagtg gaacattctt tcccattttg ttctacaaga 240

atatttttgt tattgtcttt gggctttcta tatacatttt gaaatgaggt tgacaagtta 300

cctaggaaaa ctgtcttcat aacaggccta ttgattggaa agtttgtcaa cgaattgtgg 360

gtcttttggg gtttgctgcc ccttttacgc aatgtggata tcctgcttta atgcctttat 420

atgcatgtat acaagcaaaa caggctttta ctttctcgcc aacttacaag gcctttctca 480

gtaaacagta tatgaccctt taccccgttg ctcggcaacg gcctggtctg tgccaagtgt 540

ttgctgacgc aacccccact ggttggggct tggccatagg ccatcagcgc atgcgtggaa 600

cctttgtgtc tcctctgccg atccatactg cggaactcct agccgcttgt tttgctcgca 660

gcaggtctgg agcaaacctc atcgggaccg acaattctgt cgtactctcc cgcaagtata 720

catcgtttcc atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg 780

tttacgtccc gtcggcgctg aatcccgcgg acgacccctc ccggggccgc ttggggctct 840

accgcccgct tctccgtctg ccgtaccgtc cgaccacggg gcgcacctct ctttacgcgg 900

actccccgtc tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg 960

tcgcatggag gccaccgtga acgcccaccg gaacctgccc aaggtcttgc ataagaggac 1020

tcttggactt tcagcaatgt catctggcta ataaaggaaa tttattttca ttgcaatagt 1080

gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa 1140

acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat 1200

gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc 1260

cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt 1320

atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct 1380

cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat cgaagacagt 1440

cag 1443

<210> 53

<211> 920

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 14 complete 5'

<400> 53

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatgctga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 360

ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 420

cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt 480

gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540

ggccatccac gctgttttga cctccatagt ggacaccggg accgatccag cctccgcggc 600

cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgaccctgg cagaactcgg 660

taagtctgtt gacatgtatg tgatgtatac taacctgcat gggacgtgga tttacttgtg 720

tatgtcagat agagtaaaga ttaactcttg catgtgagcg gggcatcgag atagcgataa 780

atgagtcagg aggacggata cttatatgtg ttgttatcct cctctacagt caaacagatt 840

aagcgtctca ggggagatct agcttgcttg ttctttttgc agaagctcag aataaacgct 900

caactttggc cgccaccatg 920

<210> 54

<211> 1821

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 14 complete 3'

<400> 54

ctgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60

gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120

tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180

tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240

tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300

tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360

gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420

atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480

aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540

atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600

ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660

tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720

gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780

tatacatttt gaaatgaggt tgacaagtta cctaggaaaa ctgtcttcat aatcaacctc 840

tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc 900

tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca 960

ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg 1020

tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca 1080

ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg 1140

cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 1200

acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg 1260

ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg 1320

accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcctctt cgccttcgcc 1380

ctcagacgag tcggatctcc ctttgggccg cctccccgca tctggctaat aaaggaaatt 1440

tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg 1500

gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc 1560

ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca 1620

gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt 1680

tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt 1740

actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt 1800

atggagatcg aagacagtca g 1821

<210> 55

<211> 643

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 15 complete 5'

<400> 55

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatgctga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 360

ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 420

cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt 480

gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540

ggccatccac gctgttttga cctccatagt ggacaccggg accgatccag cctccgcgtc 600

tcaggggaga tctacaccca agctgtctag agccgccacc atg 643

<210> 56

<211> 1953

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 15 complete 3'

<400> 56

ctgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60

gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120

tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180

tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240

tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300

tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360

gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420

atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480

aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540

atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600

ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660

tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720

gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780

tatacatttt gaaatgaggt tgacaagtta cctaggaaaa ctgtcttcat aacaggccta 840

ttgattggaa agtttgtcaa cgaattgtgg gtcttttggg gtttgctgcc ccttttacgc 900

aatgtggata tcctgcttta atgcctttat atgcatgtat acaagcaaaa caggctttta 960

ctttctcgcc aacttacaag gcctttctca gtaaacagta tatgaccctt taccccgttg 1020

ctcggcaacg gcctggtctg tgccaagtgt ttgctgacgc aacccccact ggttggggct 1080

tggccatagg ccatcagcgc atgcgtggaa cctttgtgtc tcctctgccg atccatactg 1140

cggaactcct agccgcttgt tttgctcgca gcaggtctgg agcaaacctc atcgggaccg 1200

acaattctgt cgtactctcc cgcaagtata catcgtttcc atggctgcta ggctgtgctg 1260

ccaactggat cctgcgcggg acgtcctttg tttacgtccc gtcggcgctg aatcccgcgg 1320

acgacccctc ccggggccgc ttggggctct accgcccgct tctccgtctg ccgtaccgtc 1380

cgaccacggg gcgcacctct ctttacgcgg actccccgtc tgtgccttct catctgccgg 1440

accgtgtgca cttcgcttca cctctgcacg tcgcatggag gccaccgtga acgcccaccg 1500

gaacctgccc aaggtcttgc ataagaggac tcttggactt tcagcaatgt catctggcta 1560

ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg 1620

aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt 1680

ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca 1740

gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg 1800

ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc 1860

cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt 1920

ccctcttctc ttatggagat cgaagacagt cag 1953

<210> 57

<211> 673

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 16 complete 5'

<400> 57

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatgctga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 360

ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 420

cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt 480

gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540

ggccatccac gctgttttga cctccatagt ggacaccggg accgatccag cctccgcgtc 600

tcaggggaga tctagcttgc ttgttctttt tgcagaagct cagaataaac gctcaacttt 660

ggccgccacc atg 673

<210> 58

<211> 1690

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 16 complete 3'

<400> 58

ctgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60

gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120

tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180

tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240

tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300

tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360

gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420

atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480

aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540

atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600

ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660

tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720

gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780

tatacatttt gaaatgaggt tgacaagtta cctaggaaaa ctgtcttcat ataatcaacc 840

tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 900

gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 960

cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 1020

tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 1080

cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 1140

ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 1200

tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 1260

tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 1320

ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcctc ttcgccttcg 1380

ccctcagacg agtcggatct ccctttgggc cgcctccccg catcatcttg ccagccatct 1440

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 1500

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 1560

ggtggggtgg ggcaggacag caagggggag gattgggaat acaatagcag gcatgctggg 1620

gatgcggtgg gctctatggg tacccaggtg ctgaagaatt gacccggttc ctcctgggga 1680

agacagtcag 1690

<210> 59

<211> 1126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 17 complete 5'

<400> 59

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accaatgacg tcgaggagaa gttccccaac tttcccgcct ctcagccttt gaaagaaaga 360

aaggggaggg ggcaggccgc gtgcagccgc gagcggtgct gggctccggc tccaattccc 420

catctcagtc gttcccaaag tcctcctgtt tcatccaagc gtgtaagggt ccccgtcctt 480

gactccctag tgtcctgctg cccacagtcc agtcctggga accagcaccg atcacctccc 540

atcgggccaa tctcagtccc ttccccccta cgtcggggcc cacacgctcg gtgcgtgccc 600

agttgaacca ggcggctgcg gaaaaaaaaa agcggggaga aagtagggcc cggctactag 660

cggttttacg ggcgcacgta gctcaggcct caagaccttg ggctgggact ggctgagcct 720

ggcgggaggc ggggtccgag tcaccgcctg ccgccgcgcc cccggtttct ataaattgag 780

cccgcagcct cccgcttcgc tctctgctcc tcctgttcga cagtcagccg catcttcttt 840

tgcgtcgcca gcctggcaga actcggtaag tctgttgaca tgtatgtgat gtatactaac 900

ctgcatggga cgtggattta cttgtgtatg tcagatagag taaagattaa ctcttgcatg 960

tgagcggggc atcgagatag cgataaatga gtcaggagga cggatactta tatgtgttgt 1020

tatcctcctc tacagtcaaa cagattaagc gtctcagggg agatctagct tgcttgttct 1080

ttttgcagaa gctcagaata aacgctcaac tttggccgcc accatg 1126

<210> 60

<211> 1126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 17 complete 3'

<400> 60

ggttcccttt tattttttac atataaatat atttccctgt ttttctaaaa aagaaaaaga 60

tcatcatttt cccattgtaa aatgccatat ttttttcata ggtcacttac atatatcaat 120

gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc cacacatctc 180

atgctttgct ctaaatcttg atatttagtg gaacattctt tcccattttg ttctacaaga 240

atatttttgt tattgtcttt gggctttcta tatacatttt gaaatgaggt tgacaagtta 300

cctaggaaaa ctgtcttcat aatcaacctc tggattacaa aatttgtgaa agattgactg 360

gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt 420

atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc 480

tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt 540

ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga 600

ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct 660

gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat 720

cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct 780

gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc 840

tgcggcctct tccgcctctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg 900

cctccccgca tcctgcccgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc 960

ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt 1020

ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag 1080

gggcccaagt tgggaagaaa cctgtagggc ctgcgaagac agtcag 1126

<210> 61

<211> 820

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 18 complete 5'

<400> 61

ctctggagac gacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180

ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240

tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300

accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 360

cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 420

ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga 480

ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg 540

cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cggcgtctca ggggagatct 600

ggtgaggtgt ggcaggcttg agatccagct gttggggtga gtactccctc tcaaaagcgg 660

gcattacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcacct 720

ggcccgatct ggccatacac ttgagtgaca atgacatcca ctttgccttt ctctccacag 780

gtgtccactc ccaggtccaa gtttaaacgc cgccaccatg 820

<210> 62

<211> 1091

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation in laboratory-polynucleotide cassette 18 complete 3'

<400> 62

ctgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60

gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120

tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180

tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240

tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300

tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360

gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420

atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480

aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540

atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600

ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660

tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720

gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780

tatacatttt gaaatgaggt tgacaagtta cctaggaaaa ctgtcttctt gccagccatc 840

tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 900

ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 960

gggtggggtg gggcaggaca gcaaggggga ggattgggaa tacaatagca ggcatgctgg 1020

ggatgcggtg ggctctatgg gtacccaggt gctgaagaat tgacccggtt cctcctgggg 1080

aagacagtca g 1091

<210> 63

<400> 63

000

<210> 64

<211> 151

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of the cassette 7 enhancer promoter linker in the laboratory

<400> 64

gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 60

ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 120

ccccatctcc cccccctccc cacccccaat t 151

<210> 65

<211> 151

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 7 promoter intron linker in laboratory

<400> 65

tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 60

cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct 120

cgcgccgccc gccccggctc tgactgaccg c 151

<210> 66

<211> 86

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of a 5' UTR linker for the intron of cassette 7 in the laboratory

<400> 66

agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattcggct 60

tgatcgaagc cgtctcaggg gagatc 86

<210> 67

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of a cassette 7 UTR Kozak linker in the laboratory

<400> 67

tttttgcaga agctcagaat aaacgctcaa ctttggccgc c 41

<210> 68

<211> 90

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 7 enhancer RNA export linker in laboratory

<400> 68

ccctgttttt ctaaaaaaga cctaggaaaa ctgtcttcat aacaggccta ttgattggaa 60

agtttgtcaa cgaattgtgg gtcttttggg 90

<210> 69

<211> 90

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 7 RNA export PolyA linker in laboratory

<400> 69

ccctgttttt ctaaaaaaga cctaggaaaa ctgtcttcat aacaggccta ttgattggaa 60

agtttgtcaa cgaattgtgg gtcttttggg 90

<210> 70

<211> 100

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of the cassette 10 enhancer promoter linker in the laboratory

<400> 70

cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac cagcacatcg 60

cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt 100

<210> 71

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 10 promoter intron linker in laboratory

<400> 71

gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg 60

gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac 120

<210> 72

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of a cassette 10 intron UTR in the laboratory

<400> 72

gacagtggtt caaagttttt ttcttccatt tcaggtgtcc tgaacacgtc tcagg 55

<210> 73

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of a cassette 10 UTR Kozak linker in the laboratory

<400> 73

agctgtctag agccg 15

<210> 74

<211> 146

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 10 enhancer RNA export linker in laboratory

<400> 74

gtctttgggc tttctatata cattttgaaa tgaggttgac aagttaccta ggaaaactgt 60

cttcatataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 120

atgttgctcc ttttacgcta tgtgga 146

<210> 75

<211> 98

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of 10-cassette RNA export PolyA linker in laboratory

<400> 75

cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatca tcttgccagc 60

catctgttgt ttgcccctcc cccgtgcctt ccttgacc 98

<210> 76

<211> 68

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of the cassette 11 TPL enhancer linker in the laboratory

<400> 76

aggcggtctt gagacggcgg atggtcgagg tgaggtgtgg caggcttgag atccagctgt 60

tggggtga 68

<210> 77

<211> 129

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 11 promoter TPL linker in laboratory

<400> 77

cgctgttttg acctccatag tggacaccgg gaccgatcca gcctccgcgt ctcaggggag 60

atctcgttta gtgaaccgtc agatcctcac tctcttccgc atcgctgtct gcgagggcca 120

gctgttggg 129

<210> 78

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 11 promoter intron linker in laboratory

<400> 78

ttgatattca cctggcccga tctggccata cacttg 36

<210> 79

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of a cassette 11 UTR Kozak linker in the laboratory

<400> 79

cccaggtcca agtttaaacg cc 22

<210> 80

<211> 77

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of the 11 enhancer PolyA linker cassette in the laboratory

<400> 80

tctttgggct ttctatatac attttgaaat gaggttgaca agttacctag gaaaactgtc 60

ttcctgcccg ggtggca 77

<210> 81

<211> 149

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 12 promoter TPL linker in laboratory

<400> 81

tcagatcgcc tggagaggcc atccacgctg ttttgacctc catagtggac accgggaccg 60

atccagcctc cgcgtctcag gggagatctc gtttagtgaa ccgtcagatc ctcactctct 120

tccgcatcgc tgtctgcgag ggccagctg 149

<210> 82

<211> 95

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of the cassette 12 TPL enhancer linker in the laboratory

<400> 82

gatgtaatta aagtaggcgg tcttgagacg gcggatggtc gaggtgaggt gtggcaggct 60

tgagatccag ctgttggggt gagtactccc tctca 95

<210> 83

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 12 enhancer intron linker in laboratory

<400> 83

gatttgatat tcacctggcc cgatctggcc atacacttga g 41

<210> 84

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of a cassette 12 UTR Kozak linker in the laboratory

<400> 84

tgtccactcc caggtccaag tttaaacgc 29

<210> 85

<211> 76

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 12 enhancer RNA export linker in laboratory

<400> 85

tgtttttcta aaaaagacct aggaaaactg tcttcataac aggcctattg attggaaagt 60

ttgtcaacga attgtg 76

<210> 86

<211> 110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 12 RNA export PolyA linker in laboratory

<400> 86

aaggtcttgc ataagaggac tcttggactt tcagcaatgt catcttgcca gccatctgtt 60

gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 110

<210> 87

<211> 110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 13 promoter enhancer linker in laboratory

<400> 87

gcggccctat aaaaagcgaa gcgcgcggcg ggcggcgtct caggggagat ctggtgaggt 60

gtggcaggct tgagatccag ctgttggggt gagtactccc tctcaaaagc 110

<210> 88

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 13 enhancer intron linker in laboratory

<400> 88

ggaggatttg atattcacct ggcccgatct ggccatacac ttga 44

<210> 89

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of Kozak linker for cassette 13 intron in laboratory

<400> 89

cctttctctc cacaggtgtc cactcccagg tccaagttta aacgccgcc 49

<210> 90

<211> 129

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 13 enhancer RNA export linker in laboratory

<400> 90

attgtctttg ggctttctat atacattttg aaatgaggtt gacaagttac ctaggaaaac 60

tgtcttcata acaggcctat tgattggaaa gtttgtcaac gaattgtggg tcttttgggg 120

tttgctgcc 129

<210> 91

<211> 110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 13 RNA export PolyA linker in laboratory

<400> 91

cctgcccaag gtcttgcata agaggactct tggactttca gcaatgtcat ctggctaata 60

aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct 110

<210> 92

<211> 94

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 14 promoter intron linker in laboratory

<400> 92

cggtgcattg gaacgcggat tccccgtgcc aagagtgacc ctggcagaac tcggtaagtc 60

tgttgacatg tatgtgatgt atactaacct gcat 94

<210> 93

<211> 112

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of Kozak linker for Inclusion in cassette 14 in laboratory

<400> 93

acttatatgt gttgttatcc tcctctacag tcaaacagat taagcgtctc aggggagatc 60

tagcttgctt gttctttttg cagaagctca gaataaacgc tcaactttgg cc 112

<210> 94

<211> 99

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 14 enhancer RNA export linker in laboratory

<400> 94

gctttctata tacattttga aatgaggttg acaagttacc taggaaaact gtcttcataa 60

tcaacctctg gattacaaaa tttgtgaaag attgactgg 99

<210> 95

<211> 100

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> preparation of cassette 14 RNA export PolyA linker in laboratory

<400> 95

cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatctggct 60

aataaaggaa atttattttc attgcaatag tgtgttggaa 100

<210> 96

<211> 280

<212> DNA

<213> hen (Gallus galllus)

<400> 96

tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc 60

caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg 120

gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt 180

gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 240

cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 280

<210> 97

<211> 973

<212> DNA

<213> hen (Gallus galllus)

<400> 97

ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg 60

ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct 120

ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa 180

agccttgagg ggctccggga gggccctttg tgcggggggg agcggctcgg ggggtgcgtg 240

cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 300

ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 360

gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 420

gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 480

cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 540

tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 600

ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 660

cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 720

gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 780

cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 840

gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 900

gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 960

gaccggcggc tct 973