RU2795471C2 - Lyophilized pharmaceutical compositions for naked dna gene therapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the production of a lyophilized pharmaceutical composition, and can be used in medicine for the treatment of neuropathy. The composition contains an effective amount of plasmid DNA expressing two isoforms of the human HGF protein, potassium phosphate buffer, mannitol, sucrose and sodium chloride.

EFFECT: invention makes it possible to obtain a lyophilized pharmaceutical composition that retains the stability of a DNA plasmid during lyophilization and pharmaceutical activity for genetic therapy after its re-dissolution in water.

11 cl, 55 dwg, 44 tbl, 4 ex

Description

1. Cross-reference to related applications

This application claims priority in US Provisional Application No. 62/700655, filed July 19, 2018, which is hereby incorporated by reference in its entirety.

2. Sequence listing

This application contains a sequence listing that was filed through the EFS-Web EFS electronic archiving file system, from English. electronic filing system) and is hereby incorporated by reference in its entirety. The specified ASCII copy created on July 16, 2019 is named 40319US_CRF_sequencelisting and has a size of 125979 bytes.

3. Prior Art

There is now substantial clinical evidence that gene therapy involving the direct in vivo delivery of nucleic acid constructs that have not been packaged in a virus or virus-like particles - so-called naked DNA or RNA constructs - can be effective in the treatment of various diseases. For example, direct intramuscular injection of a plasmid DNA construct that expresses two isoforms of the human HGF (hepatocyte growth factor) protein (i.e. pCK-HGF-X7, also referred to as "VM202") has been demonstrated to be effective in the treatment of neuropathic pain. . In a phase II clinical trial, injections of VM202 into the gastrocnemius muscle of patients with diabetic peripheral neuropathy have been shown to significantly reduce pain - two days of treatment two weeks apart was sufficient to provide symptomatic relief with improved quality of life for 3 months. Kessler et al., Annals Clin. Transl. Neurology 2(5): 465-478 (2015). The same DNA plasmid construct has also been shown to be effective in the treatment of patients with amyotrophic lateral sclerosis (ALS). In the Phase I clinical trial, a near-half of ALS patients remained stable or improved after VM202 administration with 47%, 50%, and 24% of subjects at months 1, 2, and 3, respectively, experiencing neither reduction nor improvement in ALSFRS-score. R (Amyotrophic Lateral Sclerosis Functional Rating Scale Revised), indicating physical functioning of patients with ALS that is better than that observed in historical controls. Robert L. Sufit et al., Amyotrophic Lateral Sclerosis and Frontoemporal Degeneration 18: 269-278 (2017).

However, despite the recent approval of two antisense oligonucleotide drugs for direct injection—nusinersen for intrathecal injection and eteplirsen for intravenous injection—few naked DNA or RNA constructs have actually been approved for human gene therapy. Thus, despite decades of experience in the preparation of naked nucleic acids as preparations for laboratory use, and more recent experience in the preparation of nucleic acids in the form of preparations for ex vivo gene therapy approaches, little research has been done on the preparation of DNA as a pharmaceutical product for direct therapeutic delivery. In particular, there has been little research on the stability of the nucleic acid active ingredient under different storage conditions, or on the excipients required to ensure uniform dissolution of the drug to be administered, or on ways to reduce the burden of impurities, etc. These properties are important for better and more reproducible therapeutic efficacy and safety of DNA-based drugs, as well as for the economic scalability of drug production and distribution.

Therefore, there is a need for the preparation of a naked DNA pharmaceutical drug product that provides improved stability, safety, and economic feasibility.

4. Brief summary of the invention

In one aspect, the present invention provides a novel lyophilized pharmaceutical composition that maintains the stability of a DNA construct (eg, plasmid) during lyophilization. In particular, this drug reduces the conformational change of DNA constructs during lyophilization from a more stable supercoiled form to a less stable open circular and linear form. This novel lyophilized formulation further provides uniform dissolution of the DNA constructs in a pharmaceutically acceptable solution, ensuring complete recovery of active ingredients, minimizing partial loss of potency, and ensuring that active ingredients are administered in a precise and uniform manner. In addition, the novel lyophilized preparation gives a uniform and first class appearance of the precipitate after lyophilization, thus enabling visual inspection of the quality of the drug based on DNA.

In some embodiments, this new lyophilized preparation contains plasmid DNA, where this pharmaceutical composition is obtained by lyophilization of a liquid composition, which contains before lyophilization: a. DNA of the first plasmid; b. potassium phosphate buffer with a pH in the range from 7.0 to 9.0; With. mannitol in a concentration ranging from 0 wt./about. % up to 3 wt./about. %; d. sucrose at a concentration greater than 0.5 wt./about. % and less than 1.1 wt./about. % and e. NaCl in a concentration in the range of 0.1 wt./about. % to 0.9 wt./vol. %. The first plasmid can be selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10 and pCK-SDF-1α.

In some embodiments, the first plasmid is VM202. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF- 1X10 and pCK-SDF-1α.

In some embodiments, the first plasmid is pTx-HGF-X7. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1Ec. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1X6. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1X6. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1X10. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, and pCK- SDF-1α.

In some embodiments, the first plasmid is pCK-SDF-1α. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, and pTx- IGF-1X10.

In some embodiments, at least 90% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 92.5% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 95% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 97% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 98% of the plasmid DNA in the liquid composition is supercoiled.

In some embodiments, at least 90% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 92.5% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 95% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 97% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 98% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved.

In some embodiments, at least 90% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after dissolution of the lyophilized pharmaceutical composition. In some embodiments, at least 92.5% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after dissolution of the lyophilized pharmaceutical composition. In some embodiments, at least 95% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after dissolution of the lyophilized pharmaceutical composition.

In some embodiments, at least 80% of the plasmid DNA remains supercoiled after 30 minutes after dissolution, where the lyophilized pharmaceutical composition was stored at 40° C. for 10 weeks before dissolution.

In some embodiments, the lyophilized pharmaceutical composition contains supercoiled DNA in an amount of at least 90% of the total amount of supercoiled DNA in the liquid composition. In some embodiments, the lyophilized pharmaceutical composition contains supercoiled DNA in an amount of at least 92.5% of the total amount of supercoiled DNA in the liquid composition. In some embodiments, the lyophilized pharmaceutical composition contains supercoiled DNA in an amount of at least 95% of the total amount of supercoiled DNA in the liquid composition.

In some embodiments, the liquid composition contains the first plasmid DNA at a concentration in the range of 0.1 to 1 mg/mL. In some embodiments, the liquid composition contains the first plasmid DNA at a concentration in the range of 0.25 to 0.75 mg/ml. In some embodiments, the liquid composition contains the first plasmid DNA at a concentration in the range of 0.4 to 0.6 mg/ml. In some embodiments, the liquid composition contains the first plasmid DNA at a concentration of 0.5 mg/ml.

In some embodiments, the liquid composition contains the second plasmid DNA at a concentration in the range of 0.1 to 1 mg/mL. In some embodiments, the liquid composition contains the second plasmid DNA at a concentration in the range of 0.25 to 0.75 mg/ml. In some embodiments, the liquid composition contains the second plasmid DNA at a concentration in the range of 0.4 to 0.6 mg/ml. In some embodiments, the liquid composition contains the second plasmid DNA at a concentration of 0.5 mg/ml.

In some embodiments, the liquid composition contains potassium phosphate at a concentration ranging from 5 mM to 15 mM. In some embodiments, the liquid composition contains potassium phosphate at a concentration ranging from 7.5 mM to 12.5 mM. In some embodiments, the liquid composition contains potassium phosphate at a concentration ranging from 9 mM to 11 mM. In some embodiments, the liquid composition contains potassium phosphate at a concentration of 10 mm.

In some embodiments, the liquid composition contains a potassium phosphate buffer with a pH in the range from 7.0 to 8.5. In some embodiments, the liquid composition contains a potassium phosphate buffer with a pH in the range from 7.0 to 8.0. In some embodiments, the liquid composition contains a potassium phosphate buffer with a pH of 8.0.

In some embodiments, the liquid composition contains mannitol at a concentration ranging from 1.5 wt./about. % up to 3 wt./about. %. In some embodiments, the liquid composition contains mannitol at a concentration ranging from 2 wt./about. % up to 3 wt./about. %. In some embodiments, the liquid composition contains mannitol at a concentration of 2 wt./about. %.

In some embodiments, the liquid composition contains sucrose in a concentration in the range from 0.75 wt./about. % to 1.1 wt./about. %. In some embodiments, the liquid composition contains sucrose in a concentration ranging from 0.9 wt./about. % to 1.0 wt./vol. %. In some embodiments, the liquid composition contains sucrose at a concentration of 1.0 wt./about. %.

In some embodiments, the liquid composition contains NaCl in a concentration ranging from 0.1 wt./about. % to 0.75 wt./vol. %. In some embodiments, the liquid composition contains NaCl in a concentration ranging from 0.1 wt./about. % to 0.6 wt./vol. %. In some embodiments, the liquid composition contains NaCl at a concentration of 0.4 wt./about. % to 0.5 wt./vol. %. In some embodiments, the liquid composition contains NaCl at a concentration of 0.45 wt./about. %.

Some embodiments of the present invention relate to a lyophilized pharmaceutical composition containing plasmid DNA, where this pharmaceutical composition is obtained by the implementation of lyophilization of a liquid composition, which contains before lyophilization: a. DNA of the first plasmid at a concentration of 0.5 mg/ml; b. 10 mM potassium phosphate buffer, pH 8.0; With. mannitol at a concentration of 2 wt./about. %; d. sucrose at a concentration of 1.0 wt./about. % and e. NaCl at a concentration of 0.45 wt./about. %, where at least 95% of the plasmid DNA is supercoiled and at least 90% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after dissolving the lyophilized pharmaceutical composition. The first plasmid may be selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10 and pCK-SDF-1α.

In some embodiments, the first plasmid is VM202. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF- 1X10 and pCK-SDF-1α.

In some embodiments, the first plasmid is pTx-HGF-X7. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1Ec. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1Ea. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1X6. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1X10. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, and pCK- SDF-1α.

In some embodiments, the first plasmid is pCK-SDF-1α. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, and pTx- IGF-1X10.

Some aspects of the present invention relate to a dissolved composition obtained by dissolving a lyophilized pharmaceutical composition in water.

In some embodiments, the light absorption of the dissolved composition at 450 nm is less than 0.002. In some embodiments, the light absorption of the dissolved composition at 450 nm is 0.001 or less than 0.001. In some embodiments, light absorption is measured on the day of dissolution. In some embodiments, the light absorption of the dissolved composition at 450 nm is measured after storage of the lyophilized pharmaceutical composition for 10 weeks.

Some aspects of the present invention relate to a lyophilized pharmaceutical composition containing plasmid DNA, in a single dose, where this pharmaceutical composition is obtained by the implementation of lyophilization of a liquid composition, which contains before lyophilization: a. DNA of the first plasmid at a concentration of 0.5 mg/ml; b. 10 mM potassium phosphate buffer, pH 8.0; With. mannitol at a concentration of 2 wt./about. %; d. sucrose at a concentration of 1.0 wt./about. % and e. NaCl at a concentration of 0.45 wt./about. %, where this lyophilized pharmaceutical composition is in a vial, and this vial contains a total of 2.5 mg of plasmid DNA. The first plasmid may be selected from the group consisting of VM202, pTx-HGF-X7, pTX-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10 and pCK-SDF-1α.

In some embodiments, the first plasmid is VM202. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF- 1X10 and pCK-SDF-1α.

In some embodiments, the first plasmid is pTx-HGF-X7. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1Ec. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1Ea. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1X6, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1X6. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X10, and pCK- SDF-1α.

In some embodiments, the first plasmid is pTx-IGF-1X10. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, and pCK- SDF-1α.

In some embodiments, the first plasmid is pCK-SDF-1α. In some embodiments, the lyophilized pharmaceutical composition further comprises a second plasmid DNA, wherein the second plasmid is selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, and pTx- IGF-1X10.

Some aspects of the present invention relate to a dissolved composition obtained by dissolving a lyophilized pharmaceutical composition.

Some aspects of the present invention relate to a method for obtaining a lyophilized pharmaceutical composition containing plasmid DNA, and this method includes: providing a liquid composition containing: a. DNA of the first plasmid; b. potassium phosphate buffer with a pH in the range from 7.0 to 9.0; With. mannitol in a concentration ranging from 0 wt./about. % up to 3 wt./about. %; d. sucrose at a concentration greater than 0.5 wt./about. % and less than 1.1 wt./about. % and e. NaCl in a concentration in the range of 0.1 wt./about. % to 0.9 wt./vol. %; and effecting lyophilization of the liquid composition, thereby obtaining a lyophilized pharmaceutical composition. The first plasmid may be selected from the group consisting of VM202, pTx-HGF-X7, pTX-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10 and pCK-SDF-1α.

In some embodiments, the first plasmid is VM202. In some embodiments, the first plasmid is pTx-HGF-X7. In some embodiments, the first plasmid is pTx-IGF-1X6. In some embodiments, the first plasmid is pTx-IGF-1X10. In some embodiments, the first plasmid is pTx-IGF-1Ec. In some embodiments, the first plasmid is pTx-IGF-1Ea. In some embodiments, the first plasmid is pCK-SDF-1α.

In some embodiments, the step of performing lyophilization includes: (i) loading a liquid composition; (ii) freezing; (iii) primary drying; and (iv) secondary drying.

In some embodiments, the loading step is carried out at 5°C.

In some embodiments, the freezing step is carried out at elevated temperatures in the range of -50°C to -20°C.

In some embodiments, the primary drying step is carried out at -20°C.

In some embodiments, the secondary drying step is carried out at elevated temperatures in the range of -20°C to 20°C.

In some embodiments, at least 90% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 92.5% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 95% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 97% of the plasmid DNA in the liquid composition is supercoiled.

In some embodiments, at least 90% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 92.5% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 95% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 97% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. In some embodiments, at least 98% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved.

In some embodiments, at least 90% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after dissolution of the lyophilized pharmaceutical composition. In some embodiments, at least 92.5% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after dissolution of the lyophilized pharmaceutical composition. In some embodiments, at least 95% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after dissolution of the lyophilized pharmaceutical composition.

In some embodiments, at least 80% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved, where the lyophilized pharmaceutical composition is stored at 40° C. for 10 weeks before dissolution.

In some embodiments, the lyophilized pharmaceutical composition contains supercoiled DNA in an amount of at least 90% of the total amount of supercoiled DNA in the liquid composition. In some embodiments, the lyophilized pharmaceutical composition contains supercoiled DNA in an amount of at least 92.5% of the total amount of supercoiled DNA in the liquid composition. In some embodiments, the lyophilized pharmaceutical composition contains supercoiled DNA in an amount of at least 95% of the total amount of supercoiled DNA in the liquid composition.

In some embodiments, the liquid composition contains the plasmid at a concentration in the range of 0.1 to 1 mg/mL. In some embodiments, the liquid composition contains the first plasmid at a concentration in the range of 0.25 to 0.75 mg/ml. In some embodiments, the liquid composition contains the first plasmid at a concentration in the range of 0.4 to 0.6 mg/ml. In some embodiments, the liquid composition contains the first plasmid at a concentration of 0.5 mg/ml.

In some embodiments, the liquid composition contains potassium phosphate at a concentration ranging from 5 mM to 15 mM. In some embodiments, the liquid composition contains potassium phosphate at a concentration ranging from 7.5 mM to 12.5 mM. In some embodiments, the liquid composition contains potassium phosphate at a concentration ranging from 9 mM to 11 mM. In some embodiments, the liquid composition contains potassium phosphate at a concentration of 10 mm.

In some embodiments, the liquid composition contains a potassium phosphate buffer with a pH in the range from 7.0 to 8.5. In some embodiments, the liquid composition contains a potassium phosphate buffer with a pH in the range from 7.0 to 8.0. In some embodiments, the liquid composition contains a potassium phosphate buffer with a pH of 8.0.

In some embodiments, the liquid composition contains mannitol at a concentration ranging from 1.5 wt./about. % up to 3 wt./about. %. In some embodiments, the liquid composition contains mannitol at a concentration ranging from 2 wt./about. % up to 3 wt./about. %. In some embodiments, the liquid composition contains mannitol at a concentration of 2 wt./about. %.

In some embodiments, the liquid composition contains sucrose at a concentration greater than 0.75 wt./about. % and less than 1.1 wt./about. %. In some embodiments, the liquid composition contains sucrose at a concentration greater than 0.9 wt./about. % and less than 1.1 wt./about. %. In some embodiments, the liquid composition contains sucrose at a concentration of 1.0 wt./about. %.

In some embodiments, the liquid composition contains NaCl in a concentration ranging from 0.1 wt./about. % to 0.75 wt./vol. %. In some embodiments, the liquid composition contains NaCl in a concentration ranging from 0.1 wt./about. % to 0.6 wt./vol. %. In some embodiments, the liquid composition contains NaCl in a concentration ranging from 0.4 wt./about. % to 0.5 wt./vol. %. In some embodiments, the liquid composition contains NaCl at a concentration of 0.45 wt./about. %.

Another aspect of the present invention relates to a method for treating a disease, comprising the step of: administering an effective amount of a dissolved drug to a patient with a disease, where this dissolved drug is obtained by dissolving a lyophilized pharmaceutical composition of the present invention.

In some embodiments, this method further includes the step of dissolving the lyophilized pharmaceutical composition in water, thereby obtaining a dissolved drug.

In some embodiments, the light absorption of the dissolved drug at 450 nm (A450) is less than 0.003. In some embodiments, the light absorption is less than 0.002. In some embodiments, the light absorption is 0.001 or less than 0.001.

In some embodiments, the disease is selected from the group consisting of neuropathy, ischemic disease, muscle atrophy, vascular disease, and heart disease. In some embodiments, the disease is selected from ischemic limb disease, diabetic peripheral neuropathy (DPN), amyotrophic lateral sclerosis (ALS), peripheral vascular disease, and coronary artery disease (CAD).

In some embodiments, the step of administering includes intramuscular injection of the dissolved drug.

5. Brief description of graphic materials

On FIG. 1 shows a typical result from capillary electrophoresis (CE) VM202. This result shows two peaks - one for the supercoiled form and the other for the open ring form.

On FIG. 2A-2D show near ambient DSC (differential scanning calorimetry) analysis results for the liquid state of different formulations from the 1st round of the small scale lyophilization cycle with or without an annealing step. In particular, in FIG. 2A shows the result for KP8M2SN, FIG. 2B shows the result for KP8MS3N, FIG. 2C shows the result for KP8MT3N, and FIG. 2D shows the result for control. The graphs indicate the glass transition temperature (Tg'), the eutectic melting temperature (Te), and the devitrification temperature (Td).

On FIG. Figure 3 shows the dynamics of temperature (left ordinate) and pressure (right ordinate) changes during the 1st round of the lyophilization cycle of KP8M2SN, KP8MS3N, KP8MT3N and control.

On FIG. 4A-4C are images of vials containing different formulations from round 1 of the small scale lyophilization cycle, before lyophilization (FIG. 4A), after lyophilization (FIG. 4B) and after dissolution (FIG. 4C).

On FIG. 5 shows the result from capillary electrophoresis (CE) of VM202 preparations from the 1st round of the small scale lyophilization cycle before lyophilization or after dissolution.

On FIG. 6A-6D show near ambient DSC analysis results for the liquid state of various preparations from the 2nd round of the small scale lyophilization cycle with or without an annealing step. In particular, in FIG. 6A shows the result for 4MSN, FIG. 6B shows the result for 3MSN, FIG. 6C shows the result for 2MSN, and FIG. 6D shows the result for control.

On FIG. Figure 7 shows temperature (left ordinate) and pressure (right ordinate) trends during round 2 of the lyophilization cycle of the first set of 4MSN, 3MSN, 2MSN, and controls.

On FIG. 8A-8C are images of vials containing the first set of 4MSN, 3MSN, 2MSN and controls from the 2nd round of the lyophilization cycle on a small scale, before lyophilization (Fig. 8A), after lyophilization (Fig. 8B) and after dissolution (Fig. 8C). ).

On FIG. 9 shows the result from capillary electrophoresis (CE) of the first set of 4MSN, 3MSN, 2MSN and control from the second 2nd round of the small scale lyophilization cycle before lyophilization or after dissolution.

On FIG. 10 shows temperature (left ordinate) and pressure (right ordinate) trends during round 2 of the lyophilization cycle of the second set of 4MSN, 3MSN, 2MSN, and controls.

On FIG. 11A-11B are images of vials containing the second set of 4MSN, 3MSN, 2MSN and control from the second 2nd round of the small scale lyophilization cycle before lyophilization (FIG. 11A) and after lyophilization (FIG. 11B). On FIG. 11C-11F show images of vials containing the second set of 4MSN (Fig. 11C), 3MSN (Fig. 11D), 2MSN (Fig. 11E) and control (Fig. 11F) after reconstitution.

On FIG. 12 shows the result from capillary electrophoresis (CE) of the second set of 4MSN, 3MSN, 2MSN and control from the second 2nd round of the small scale lyophilization cycle before lyophilization or after dissolution.

On FIG. 13 shows the dynamics of temperature (left ordinate) and pressure (right ordinate) during the 2MSN and 2M1SN lyophilization cycle.

On FIG. 14A-14B are views of vials containing 2MSN and 2M1SN before lyophilization (FIG. 14A) and after lyophilization (FIG. 14B). On FIG. 14C-14D show images of vials containing 2MSN (FIG. 14C) and 2M1SN (FIG. 14D) after dissolution.

On FIG. 15 shows the result from capillary electrophoresis (CE) of 2MSN and 2M1SN before lyophilization or after dissolution.

On FIG. 16A-16B are images of vials containing 4MSN, 3MSN, 2MSN and control after storage at 25°C for 3 days (Fig. 16A) or 7 days (Fig. 16B) after dissolution in T is 0.

On FIG. 17A-17B show the results from capillary electrophoresis (CE) 4MSN, 3MSN, 2MSN and control after storage at 25°C for 3 days (FIG. 17A) or 7 days (FIG. 17B).

On FIG. 18A-18C are images of vials containing 4MSN, 3MSN, 2MSN and control before and after dissolution of the lyophilized preparation stored for 10 weeks at 25°C (Fig. 18A), at 40°C (Fig. 18B) or at 5° C (Fig. 18C).

On FIG. 19 shows the result from capillary electrophoresis (CE) 4MSN, 3MSN, 2MSN and control after storage at 40°C for 10 weeks.

On FIG. 20A-20C are plots of supercoiled DNA percentages measured from capillary electrophoresis (CE) results for 4MSN, 3MSN, 2MSN and controls when these preparations are stored at 5°C (FIG. 20A), 25°C (FIG. 20B). or 40°C (Fig. 20C).

On FIG. 21A-21C are plots of percentages of open cDNA measured from capillary electrophoresis (CE) results for 4MSN, 3MSN, 2MSN and control while storing these preparations at 5°C (FIG. 21A), 25°C (FIG. 21B). ) or 40°C (Fig. 21C).

On FIG. 22A-22B are images of vials containing 2MSN (FIG. 22A) or 2M1SN (FIG. 22B) stored at 25°C for 3 days after dissolution. On FIG. 22C-22D are images of vials containing 2MSN (FIG. 22C) or 2M1SN (FIG. 22D) stored at 25°C for 7 days after dissolution.

On FIG. 23A-23B show the results from capillary electrophoresis (CE) of 2MSN and 2M1SN after storage at 25°C for 3 days (FIG. 23A) or 7 days (FIG. 23B).

On FIG. 24A-24C are views of vials containing 2MSN or 2M1SN before or after dissolution of the lyophilized formulation stored for 10 weeks at 25°C (FIG. 24A), at 40°C (FIG. 24B), or at 5°C (FIG. 24C).

On FIG. 25 shows the result from capillary electrophoresis (CE) of 2MSN and 2M1SN after storage at 40°C for 10 weeks.

On FIG. 26A-26C are plots of supercoiled DNA percentages measured from capillary electrophoresis (CE) results for 2MSN and 2M1SN when these preparations were stored at 5°C (FIG. 26A), 25°C (FIG. 26B), or 40°C. (Fig. 26C).

On FIG. 27A-27C are plots of open cDNA percentages measured from capillary electrophoresis (CE) results for 2MSN and 2M1SN when these preparations were stored at 5°C (FIG. 27A), 25°C (FIG. 27B), or 40°C. C (Fig. 27C).

On FIG. 28 shows the trend in temperature (left ordinate) and pressure (right ordinate) during the lyophilization cycle used in Example 2.

On FIG. 29 shows images of vials containing F1 of Example 2 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 30 shows images of vials containing F2 of Example 2 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 31 shows images of vials containing F3 of Example 2 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 32 shows images of vials containing F4 of Example 2 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 33 shows images of vials containing F5 of Example 2 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 34 shows images of vials containing F6 of Example 2 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 35 shows the result of capillary electrophoresis (CE) F1, F2, F3, F4 and F5 of Example 2.

On FIG. 36 shows images of vials containing F1 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 37 shows images of vials containing F2 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 38 shows images of vials containing F3 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 39 shows images of vials containing F4 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 40 shows images of vials containing F5 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 41 shows images of vials containing F6 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 42 shows images of vials containing F7 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 43 shows images of vials containing F8 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 44 shows images of vials containing F9 of Example 3 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 45 shows the result of capillary electrophoresis (CE) F1, F2, F3, F4, F5, F6, F7, F8 and F9 of Example 3 (TABLE 40).

On FIG. 46 shows images of vials containing F1 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 47 shows images of vials containing F2 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 48 shows images of vials containing F3 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 49 shows images of vials containing F4 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 50 shows images of vials containing F5 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 51 shows images of vials containing F6 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 52 shows images of vials containing F7 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 53 shows images of vials containing F8 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 54 shows images of vials containing F9 of Example 4 before or after dissolution of the lyophilized preparation against a white background (left) or a black background (right).

On FIG. 55 shows the result of capillary electrophoresis (CE) F1, F2, F3, F4, F5, F6, F7, F8 and F9 of Example 4 (TABLE 44).

On the data of Fig. various embodiments of the present invention are described for purposes of illustration only. One skilled in the art will readily appreciate from the following discussion that alternative embodiments of the structures and methods illustrated herein can be used without departing from the principles of the invention described herein.

6. Detailed description of the invention

6.1 Definitions

Unless otherwise defined, all technical and scientific terms used in this document have the meaning generally understood by a person skilled in the art to which this invention belongs. The following terms, as used in this document, have the meanings assigned to them below.

The term "liquid composition" as used herein refers to a composition in liquid form that contains plasmid DNA and at least one pharmaceutically acceptable excipient, and which can be lyophilized to obtain a lyophilized pharmaceutical composition as described in this document.

The term "lyophilized composition" or "lyophilized pharmaceutical composition" as used herein refers to any composition or dry form pharmaceutical composition that is obtained by lyophilization. The terms "performing lyophilization" or "lyophilization" have the meanings understood by those skilled in the art, referring broadly to any method of freezing followed by dehydration in the frozen state under vacuum. Lyophilized compositions may be dissolved for injection.

The term "dissolved" or "dissolution" as used herein refers to the restoration of the original form of a substance previously modified for conservation and storage, such as rehydration, i.e. restoring the liquid state of a plasmid DNA preparation that was previously lyophilized and stored. The lyophilized composition of the present invention can be dissolved in any aqueous solution that yields a stable solution suitable for pharmaceutical administration. Such aqueous solutions include, but are not limited to, sterile water, Tris-EDTA (TE), phosphate buffered saline (PBS), Tris buffer, and normal saline.

The term "isolated" or "biologically pure" as used herein refers to a substance that is substantially free of the components that normally accompany the substance in its natural state. Thus, isolated plasmid DNA as used herein is substantially free of components normally associated with plasmid DNA in its in situ environment, such as bacterial proteins, lipids, or cell wall components.

The term "VM202" as used herein refers to plasmid DNA, also referred to as pCK-HGF-X7, containing the vector pCK (SEQ ID NO 5) and HGF-X7 (SEQ ID NO 13) cloned into the pCK vector. VM202 was deposited under the terms of the Budapest Agreement at the Korea Microorganism Culture Center (KCCM) under accession number KCCM-10361 on March 12, 2002.

The term "HGF isoforms" as used herein refers to a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of a naturally occurring HGF polypeptide in an animal. The term includes polypeptides having an amino acid sequence that is at least 80% identical to any full-length wild-type HGF polypeptide, and includes polypeptides having an amino acid sequence that is at least 80% identical to a naturally occurring allelic HGF variant, variant , resulting from splicing, or a variant resulting from a deletion. HGF isoforms preferred for use in the present invention include two or more isoforms selected from the group consisting of full-length HGF (fIGF) (synonymous with fHGF), deletion variant HGF (dHGF), NK1, NK2 and NK4. According to a more preferred embodiment of the present invention, the HGF isoforms used in the methods described herein include fIHGF (SEQ ID NO 1) and dHGF (SEQ ID NO 2).

The terms "human flHGF", "flHGF" and "fHGF" are used interchangeably herein to refer to a protein consisting of amino acids 1-728 of the human HGF protein. The sequence of fIHGF is given in SEQ ID NO 1.

The terms "human dHGF" and "dHGF" are used interchangeably herein to refer to a deletion variant of the HGF protein produced by alternative splicing of the human HGH gene. In particular, "human dHGF" or "dHGF" refers to a human HGF protein with a five amino acid deletion (F, L, P, S and S) in the first alpha chain double loop domain of the full length HGF sequence. Human dHGF is 723 amino acids in length. The amino acid sequence of human dHGF is given in SEQ ID NO 2.

The term "IGF-1 isoform (insulin-like growth factor-1)" or "human IGF-1 isoform" as used herein refers to a polypeptide having an amino acid sequence that is at least 80 % is identical to the amino acid sequence of one of the naturally occurring human pre-pro-IGF1 polypeptides or an allelic, splicing, or deletion variant thereof. Naturally occurring pre-pro-IGF1 polypeptides include Class I isoforms, Ec (SEQ ID NO 25); Class II, Ea (SEQ ID NO 27); Class I, Eb (SEQ ID NO 29) and Class I, Ea (SEQ ID NO 23).

The terms "Isoform #1," "Class I, Ec isoform," "Class I, IGF-1Ec isoform," or "Class I, IGF-1Ec" are used interchangeably herein to name the polypeptide of SEQ ID NO 25.

The terms "Isoform #2," "Class II, Ea isoform," "Class II, IGF-1 Ea isoform," or "Class II, IGF-1 Ea" are used interchangeably herein to name the polypeptide of SEQ ID NO 27.

The terms "Isoform #3," "Class I, Eb isoform," "Class I, IGF-1 Eb isoform," or "Class I, IGF-1 Eb" are used interchangeably herein to name the polypeptide of SEQ ID NO 29.

The terms "Isoform #4," "Class I, Ea isoform," "Class I, IGF-1 Ea isoform," or "Class I, IGF-1 Ea" are used interchangeably herein to name the polypeptide of SEQ ID NO 23.

The term "treatment" as used herein refers to at least one of the following: (a) suppression of a symptom of a disease; (b) alleviating a symptom of the disease; and (c) eliminating the symptom of the disease.

In some embodiments, the composition of the present invention may treat a symptom associated with neuropathy, coronary disease, muscle atrophy, or heart disease.

The term "therapeutically effective dose" or "effective amount" as used herein refers to the dose or amount that produces the desired effect for which it is administered. In the context of the present methods, a therapeutically effective amount is an effective amount to treat a symptom of a disease. The exact dose or amount will depend on the goal of treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

The term "sufficient amount" as used herein refers to a sufficient amount to obtain the desired effect.

The term "degenerate sequence" as used herein refers to a nucleic acid sequence that can be translated to produce an identical amino acid sequence relative to a sequence translated from a reference nucleic acid sequence.

6.2. Other interpreted conventions

It is understood that the ranges listed herein are shorthand for all values within a given range, including the listed endpoints. For example, it is understood that the range from 1 to 50 includes any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25.26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.

Unless otherwise indicated, reference to a compound that has one or more stereocenters is intended to include each stereoisomer and all combinations of its stereoisomers.

6.3. Lyophilized pharmaceutical composition

In a first aspect, a lyophilized pharmaceutical composition is provided. This lyophilized pharmaceutical composition contains plasmid DNA and is obtained by lyophilization of a liquid composition which, prior to lyophilization, contains:

a. plasmid DNA of the first plasmid;

b. potassium phosphate buffer pH 7.0 to 9.0;

c. mannitol in a concentration of 0 wt./about. % up to 3 wt./about. %;

d. sucrose at a concentration greater than 0.5 wt./about. % and less than 1.1 wt./about. %; And

e. NaCl in a concentration of 0.1 wt./about. % to 0.9 wt./vol. %.

This first plasmid may be selected from the group consisting of VM202, pTx-HGF-X7, pTx-IGF-1Ec, pTx-IGF-1Ea, pTx-IGF-1X6, pTx-IGF-1X10 and pCK-SDF-1α.

6.3.1. Plasmid DNA

This liquid composition includes plasmid DNA, which is the active ingredient of the pharmaceutical composition. This plasmid DNA may include genetic material for gene therapy. In particular, this plasmid DNA may encode a genetic product that may correct the function of a defective gene or transcript, or encode polypeptides, sense or antisense oligonucleotides, or RNA (coding or non-coding; e.g., siRNA (small interfering RNA), shRNA (short hairpin RNA) , miRNAs, and their antisense equivalents (eg, antagonMiR)). A formulation containing any plasmid DNA known in the art for use in gene therapy falls within the scope of the present invention.

This liquid composition may contain plasmid DNA at a concentration that allows the subsequent dissolution of the lyophilized pharmaceutical composition to provide an effective concentration for therapeutic use of the dissolved drug. The concentration of plasmid DNA may be adjusted depending on various factors including the amount of the composition to be delivered, the diseases to be treated, the age and weight of the subject, the method of delivery and route of administration, etc.

In particular, this liquid composition may include plasmid DNA at a concentration of 0.1 to 5 mg/ml, 0.1 to 3 mg/ml, 0.1 to 2 mg/ml, 0.1 to 1 mg/ml. ml, from 0.25 to 0.75 mg/ml, from 0.4 to 0.6 mg/ml or at a concentration of 0.5 mg/ml.

This plasmid DNA may be a polynucleotide of 3000 to 15000 base pairs, 3000 to 10000 base pairs, 3000 to 9000 base pairs, 3000 to 8000 base pairs, 3000 to 7000 base pairs, 3000 to 6000 base pairs, 3000 to 5000 bp, 4000 to 8000 bp, 4000 to 7500 bp, 4000 to 6000 bp, 6000 to 9000 bp, or 7000 to 8000 bp. This plasmid DNA may be a polynucleotide of length that falls within the scope proposed herein.

6.3.1.1. vector

Plasmid DNA used in the methods of the present invention typically contains a vector with one or more regulatory sequences (eg, a promoter or enhancer) operably linked to the sequences to be expressed. This regulatory sequence regulates the expression of a protein (eg, one or more isoforms of HGF or IGF-1).

It is preferred that the polynucleotide encoding the protein is operably linked to a promoter in the expression construct. The term "operably linked" refers to a functional relationship between a nucleic acid expression control sequence (such as a promoter, a signal sequence, or a transcription factor binding site chip) and a second nucleic acid sequence, where the expression control sequence affects transcription and/or translation of the nucleic acid. corresponding to the second sequence.

In exemplary embodiments, a promoter linked to a polynucleotide is functional, preferably in animal cells, more preferably in mammalian cells, to control transcription of the polynucleotide, including promoters derived from the genome of mammalian cells or from mammalian viruses, e.g., CMV (cytomegalovirus) promoter, late promoter adenovirus, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter (herpes simplex virus), RSV promoter (Rous sarcoma virus), elongation factor 1-alpha (EFI alpha), metallothionein promoter, beta-actin promoter, human gene promoter IL-2 (interleukin-2), human IFN (interferon) gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, and human GM-CSF (granulocyte-macrophage colony stimulating factor) gene promoter, but not limited to. More preferably, the promoter useful in this invention is a promoter derived from the human CMV IE (hCMV) gene or an EFI alpha promoter, most preferably a promoter/enhancer derived from the hCMV IE gene, and a 5'-UTR (untranslated region) , containing the entire sequence of the exon 1 and exon 2 sequence, spanning the sequence immediately before the start ATG codon.

The expression cassette used in the present invention may contain a polyadenylation sequence, for example, including a bovine growth hormone terminator (Gimmi, E.R., et al., Nucleic Acids Res. 17: 6983-6998 (1989)), a polyadenylation sequence occurring from SV40 (Schek, N, et al., Mol. Cell Biol. 12: 5386-5393 (1992)), HIV-1 polyA (human immunodeficiency virus) (Klasens, B.I.F., et al., Nucleic Acids Res. 26: 1870-1876 (1998)), β-globin polyA (Gil, A., et al, Cell 49: 399-406 (1987)), TK HSV polyA (Cole, C. N. and T. P. Stacy, Mol. Cell. 5 Biol. 5: 2104-2113 (1985)) or polyA polyoma virus (Batt, D. Band G. G. Carmichael, Mol. Cell. Biol. 15: 4783-4790 (1995)), but not limited to them.

In presently preferred embodiments, the vector is pCK, pCP, pVAXI, pTX, or pCY. In particularly preferred embodiments, the vector is pCK, details of which can be found in WO 2000/040737 and Lee et al., Biochem. Biophys. Res. Comm. 272: 230-235 (2000), both of which are incorporated herein by reference in their entirety. E. coli transformed with pCK (Top10-pCK) was deposited with the Korea Microorganism Culture Center (KCCM) under the terms of the Budapest Agreement on March 21, 2003 (Access No: KCCM-10476). E. coli transformed with pCK-VEGF165 (i.e., the pCK vector with the coding sequence for VEGF (vascular endothelial growth factor) - Top10-pCK/VEGF165') was deposited at the Korea Microorganism Culture Center (KCCM) under the terms of the Budapest Agreement on December 27, 1999 (access no.: KCCM-10179).

The pCK vector is constructed such that expression of a gene, eg the HGF gene or the IGF-1 gene, is regulated under the human cytomegalovirus (HCMV) enhancer/promoter, as detailed in Lee et al., Biochem. Biophys. Res. commun. 272: 230 (2000); WO 2000/040737, which are both incorporated by reference in their entirety. The pCK vector has been used for human clinical trials and has been confirmed to be safe and effective (Henry et al., Gene Ther. 18:788 (2011)).

In other preferred embodiments, the vector is pTX (SEQ ID NO 15), a plasmid vector derived from pCK. pTX was generated by two consecutive cycles of pCK mutagenesis. A first deletion mutagenesis campaign was performed to remove an unnecessary sequence between the kanamycin resistance gene and ColE1 pCK. In particular, deletion PCR mutagenesis was performed using the first pair of primers (SEQ ID NOs: 17 and 18). A 228 bp deletion between the kanamycin resistance gene and ColE1 was confirmed by plasmid sequencing. A second deletion mutagenesis campaign was then performed using a second pair of primers (SEQ ID NOs: 19 and 20) to optimize the size of the HCMV intron sequence. The HCMV intron sequence (421 bp) between exon 1 and exon 2 of IE1 was deleted and the deletion was confirmed by sequencing.

6.3.1.2. plasmid DNA encoding human HGF

In some embodiments, the plasmid DNA encodes human HGF or a variant thereof.

Hepatocyte growth factor (HGF) is a heparin-binding glycoprotein, also known as scatter factor or hepatopoietin-A. The endogenous gene encoding human HGF is located on chromosome 7q21.1 and contains 18 exons and 17 introns (Seki T., et al., Gene 102: 213-219 (1991)). The transcript is about 6 kb. transcribed from the HGF gene, and then from it the HGF precursor polypeptide (fIHGF), consisting of 728 amino acids, is synthesized. Simultaneously, the 723 amino acid dHGF precursor polypeptide is also synthesized by alternative splicing of the HGF gene. Biologically inactive precursors can be converted to active forms of the disulfide-linked heterodimer via a serum protease. In these heterodimers, the high molecular weight alpha chain forms four double loop domains and an N-terminal hairpin like the pre-activation peptide region of plasminogen. The "double loop" domains of the triple disulfide-linked loop structure of about 80 amino acids may play an important role in protein-protein interaction. The low molecular weight beta chain forms an inactive domain similar to a serine protease. dHGF, consisting of 723 amino acids, is a polypeptide with a deletion of five amino acids in the 1st domain "double loop" of the alpha chain, i.e. F, L, P, S and S.

HGF has various biological functions such as 1) inducing epithelial cells into a tubular structure; 2) stimulation of vascularization from endothelial cells in vitro and in vivo; 3) regeneration of the liver and kidney, due to its anti-apoptotic activity; 4) organogenesis of the kidney, ovary and testis; 5) ensuring the control of osteogenesis; 6) stimulation of growth and differentiation of erythroid hematopoietic progenitor cells; and 7) neuronal axon sprouting (Stella, M.C. and Comoglio, P.M., The International Journal of Biochemistry & Cell Biology 31: 1357-1362 (1999)). Based on these different functions of HGF, either a gene encoding HGF or a variant thereof can be developed as a therapeutic agent.

Indeed, plasmids encoding one or more isoforms of human HGF have been developed and used to treat various diseases as described in US Pat. in their entirety. These plasmids can be used in various embodiments of the present disclosure.

In particular, a given plasmid can express two or more HGF isoforms by including an expression control sequence for each isoform coding sequence (CDS). In some embodiments, this construct contains an internal ribosomal entry site (IRES) between two coding sequences, for example, in the following order: (1) expression regulation sequence - (2) first isoform coding sequence - (3) IRES - (4) coding sequence of the second isoform - (5) transcription termination sequence. The IRES provides for the start of translation in the IRES sequence, thereby allowing the expression of two genes of interest from a single construct. In other embodiments, a number of constructs are used together to induce expression of more than one HGF isoform in a subject to be administered, each encoding a single HGF isoform.

In preferred embodiments of these methods, a construct is used that simultaneously expresses two or more different types of HGF isoforms—i.e. fIHGF and dHGF by including an alternative splicing site. It was previously shown in US Pat. encoding one isoform of HGF (either fIHGF or dHGF). In exemplary embodiments, this construct comprises (i) a first sequence containing exons 1-4 of the human HGF gene (SEQ ID NO 3), or a degenerate sequence of the first sequence; (ii) a second sequence containing intron 4 of the human HGF gene (SEQ ID NO 6) or a fragment of the second sequence; and (iii) a third sequence containing exons 5-18 of the human HGF gene (SEQ ID NO 4), or a degenerate sequence of this third sequence. From this construct, two isoforms of HGF (fIHGF and dHGF) can be generated by alternative splicing between exon 4 and exon 5.

In some embodiments, this construct contains the entire sequence of intron 4. In some embodiments, this construct contains a fragment of intron 4. In preferred embodiments, this construct contains a nucleotide sequence selected from the group consisting of SEQ ID NO 7 - SEQ ID NO 14. Nucleotide sequence of SEQ ID NO 7 corresponds to 7113 b.p. a polynucleotide encoding fIHGF and dHGF and including the entire sequence of intron 4. The nucleotide sequences of SEQ ID NO: 8-14 correspond to polynucleotides encoding fIHGF and dHGF and including different fragments of intron 4.

Various nucleic acid constructs containing cDNA corresponding to exons 1-18 of human HGF and intron 4 of the human HGF gene, or fragments thereof, are referred to as "HGF-X", followed by a unique number, as described in US Pat. No. 7,812,146. HGF-X analyzed by Applicant include HGF-X1 (SEQ ID NO 7), HGF-X2 (SEQ ID NO 8), HGF-X3 (SEQ ID NO 9), HGF-X4 (SEQ ID NO 10), HGF- X5 (SEQ ID NO 11), HGF-X6 (SEQ ID NO 12), HGF-X7 (SEQ ID NO 13), and HGF-X8 (SEQ ID NO 14), but are not limited to.

It has previously been demonstrated that two HGF isoforms (ie, fIHGF and dHGF) can be generated by alternative splicing between exon 4 and exon 5 of each of the constructs. In addition, among the different HGF constructs, HGF-X7 showed the highest level of expression of two HGF isoforms (i.e., fIHGF and dHGF), as disclosed in US Pat. No. 7,812,146, incorporated herein by reference in its entirety. Accordingly, a nucleic acid construct containing HGF-X7 can be used in preferred embodiments of the methods of the present invention.

In particularly preferred embodiments, a pCK plasmid containing HGF-X7 expression sequences is used as a nucleic acid construct in the methods of the present invention. In particular, pCK-HGF-X7 (also referred to as "VM202") can be used. pCK-HGF-X7 is a construct containing pCK vector (SEQ ID NO 5) and HGF-X7 (SEQ ID NO 13) cloned into pCK vector. pCK-HGF-X7 was deposited under the terms of the Budapest Agreement at the Korea Microorganism Culture Center (KCCM) under accession number KCCM-10361 on March 12, 2002.

Amino acid sequences and nucleotide sequences of HGF isoforms used in the methods described herein may further include amino acid sequences and nucleotide sequences substantially identical to those of wild-type human HGF isoforms. Substantial identity includes sequences with at least 80% identity, more preferably at least 90% identity, and most preferably at least 95% identity, wherein the amino acid sequence or nucleotide sequence of the wild-type human HGF isoform aligns with the sequence in the maximum way. Methods for aligning sequences for comparison are well known in the art. In particular, the alignment algorithm disclosed on the NCBI Basic Local Alignment Search Tool (BLAST) website of the National Center for Biotechnological Information (NBCI) and used by in connection with the sequence analysis programs blastp, blasm, blastx, tblastn, and tblastx, can be used to determine percent identity.

6.3.1.3. plasmid DNA encoding IGF-1

In some embodiments, the plasmid DNA encodes human IGF-1 or a variant thereof.

Insulin-like growth factor 1 (IGF-1) is a hormone similar in molecular structure to insulin that plays an important role in childhood growth and has anabolic effects in adults. The human IGF-1 gene contains six exons (exons 1, 2, 3, 4, 5 and 6 (6-1 and 6-2)) spanning almost 90 kb. genomic DNA. Exons 1 and 2 are mutually exclusive leader exons, with each having multiple promoter sites that are used differently. In addition, the IGF-1 gene can be differentially spliced to create multiple transcript variants. Each transcript variant encodes a different pre-pro-IGF-1 protein ("IGF-1 isoform") with variable signal peptide leader sequences. However, all transcript isoforms result in the same IGF-1 70-amino acid peptide that uses the same receptor after processing.

Pre-pro-IGF-1 peptides differ in their leader or signal sequences and in their carboxy (C) terminus. The inclusion of exon 1 or exon 2 is mutually exclusive, and one of them serves as the leader sequence of the pre-pro-IGF-1 peptide; different leader exons create different 5'-UTRs. Pre-pro-IGF-1 polypeptides undergo post-transcriptional proteolytic cleavage to remove the leader and carboxy terminus of the E-peptide, resulting in mature 70 amino acid IGF-1.

Transcripts containing exon 1 are referred to as Class 1 transcripts (eg, Class I, Ec; Class II, Eb and Class I, Ea), while transcripts containing exon 2 are referred to as Class 2 transcripts (eg, Class II, Ea). Nearly all pre-propeptides include a 27 amino acid signal peptide derived from exon 3, with the remaining signal sequences derived from the inclusion of exon 1 or 2. A minority of transcripts use a different transcription initiation site within exon 3, generating a shorter signal peptide of 22 amino acids . Exons 3 and 4 are unchanged and encode domains B, C, A and D of the mature IGF-1 peptide; exon 4 encodes two-thirds of the mature IGF-1 peptide. The human Eb peptide consists of exons 4 and 5 only, while Ec contains exons 4, 5 and 6.

Alternative splicing and mutually exclusive transcription initiation results in different pre-pro-IGF-1 polypeptides (ie, IGF-1 isoforms). In particular, the isoform of IGF-1 Class I, Ec (SEQ ID NO 25) containing at least a fragment of exons 1, 3/4, 5 and 6, is generated from a transcript containing the sequence of SEQ ID NO 26. II, Ea (SEQ ID NO 27), containing at least a fragment of exons 2, 3/4 and 6, is generated from a transcript containing the sequence of SEQ ID NO 28. IGF-1 Class I isoform, Eb (SEQ ID NO 29), containing at least a fragment of exons 1, 3/4 and 5, is generated from a transcript containing the sequence of SEQ ID NO 30. The isoform of IGF-1 Class I, Ea (SEQ ID NO 23), containing at least a fragment of exons 1, 3/ 4 and 6 is generated from a transcript containing the sequence SEQ ID NO 24.

Although the mature IGF-1 protein derived from different transcripts does not differ, it was assumed that different transcript isoforms have different regulatory functions. Form variants have different stabilities, binding partners, and activities, indicating a key regulatory role for these isoforms. The biological significance of these isoforms remains unclear, although Class I isoforms with exon 1 have been hypothesized to be autocrine/paracrine forms, while class II isoforms with exon 2 are secreted endocrine forms. This is based on the finding that Class II transcripts include a typical signal peptide motif associated with efficient secretion, while Class I transcripts have a longer signal peptide that may possibly interfere with secretion.

Plasmids encoding one or more isoforms of human IGF-1 have been designed and analyzed for the treatment of neuropathy as described in US Applications No. 16/513560 and/or 16/513564, which are incorporated herein by reference in their entirety. These plasmids can be used in various embodiments of the present disclosure.

In particular, in some embodiments, this plasmid contains the coding sequence for one of the isoforms of IGF-1. For example, the DNA construct may contain a sequence encoding Class I, Ea (SEQ ID NO 24); Class I, Eb (SEQ ID NO 30); Class I, Ec (SEQ ID NO 26) or Class II, Ea (SEQ ID NO 28).

In some embodiments, the DNA construct is a dual expression construct - a DNA construct that expresses more than one isoform of IGF-1 by including an expression control sequence for each isoform's coding sequence (CDS). In some embodiments, this construct contains an internal ribosome entry site (IRES) between two coding sequences, for example, in the following order: (1) expression regulation sequence - (2) first isoform coding sequence - (3) IRES - (4) coding sequence second isoform - (5) transcription termination sequence. The IRES provides for the start of translation in the IRES sequence, thereby allowing the expression of two protein products from a single transcript. In other embodiments, a number of constructs are used together to induce expression of more than one isoform of IGF-1 in a subject to whom an administration is made, each encoding one isoform of IGF-1.

In preferred embodiments, the DNA construct is capable of simultaneously expressing two or more of the following isoforms of IGF-1: for example, (i) Ec isoform, Class I (isoform #1) and Ea isoform, Class II (isoform #2); (ii) Ec isoform, Class I (isoform #1) and Eb isoform, Class I (isoform #3); (iii) isoform Ec, Class I (isoform #1) and isoform Ea, Class I (isoform #4); (iv) isoform Ea, Class II (isoform No. 2) and isoform Eb, Class I (isoform No. 3); (iv) isoform Ea, Class II (isoform No. 2) and isoform Eb, Class I (isoform No. 3); (v) isoform Ea, Class II (isoform No. 2) and isoform Ea, Class I (isoform No. 4); (vi) Eb isoform, Class I (isoform No. 3) and Ea isoform, Class I (isoform No. 4) by containing an alternative splicing site.

For example, the DNA construct may comprise (i) a first sequence containing exons 1, 3 and 4 of the human IGF-1 gene (SEQ ID NO 31), or a degenerate sequence of the first sequence; (ii) a second sequence containing intron 4 of the human IGF-1 gene (SEQ ID NO 32), or a fragment of this second sequence; (iii) a third sequence containing exons 5 and 6-1 of the human IGF-1 gene (SEQ ID NO 33), or a degenerate sequence of this third sequence; (iv) a fourth sequence containing intron 5 of the human IGF-1 gene (SEQ ID NO 34), or a fragment of this second sequence; and (v) a fifth sequence containing exon 6-2 of the human IGF-1 gene (SEQ ID NO 35), or a degenerate sequence of this fifth sequence. Introns 4 and 5 can undergo alternative splicing resulting in two isoforms of IGF-1 (eg, Class I, Ec and Class I, Ea).

In some embodiments, the DNA construct is tested in vitro and/or in vivo for its ability to express one or more isoforms of IGF-1. In preferred embodiments, DNA constructs are selected that are capable of expressing both IGF-1 isoforms, Class I, Ec and Class I, Ea.

Miscellaneous DNA constructs containing cDNA corresponding to (i) exons 1-6 of the human IGF-1 gene and (ii) introns 4 and 5 of the human IGF-1 gene or different fragments of introns 4 and 5 are referred to as "IGF-1X", followed by unique number. The IGF-1X constructs analyzed by Applicant include IGF-1X1, IGF-1X2, IGF-1X3, IGF-1X4, IGF-1X5, IGF-1X6, IGF-1X7, IGF-1X8, IGF-1X9 and IGF-1X10, but are not limited to them. Among the constructs analyzed, both IGF-1X6 and IGF-1X10 were identified to express IGF-1 isoforms of both Class I, Ec, and Class I, Ea.

In preferred embodiments, IGF-1X6 (SEQ ID NO 21) or IGF-1X10 (SEQ ID NO 22) is used. IGF-1X6 (SEQ ID NO 21) and IGF-1X10 (SEQ ID NO 22) cloned into the pCK vector are called pCK-IGF-1X6 and pCK-IGF-1X10, respectively. E. coli cells transformed with pCK-IGF-1X6 ("DH5α_pCK-IGF1 X6") were deposited under the terms of the Budapest Agreement at the Korea Type Culture Collection (KCTC, Korea Research Institute of Bioscience and Biotechnology (KRIBB) 181, Ipsing-gil, Jeongeup -si, Jeollabuk-do, 56212, Republic of Korea) under accession number KCTC 13539 BP on May 30, 2018. E. coli cells transformed with pCK-IGF-1X10 ("DH5α_pCK-IGF1 X10") were deposited under the terms of the Budapest Agreement in the Korean type culture collections (KCTC, Korea Research Institute of Bioscience and Biotechnology (KRIBB) 181, Ipsing-gil, Jeongeup-si, Jeollabuk-do, 56212, Republic of Korea) accession number KCTC 13540 BP 30 May 2018.

In some embodiments, the pTX plasmid contains IGF-1X6 (ie pTx-IGF-1X6) or IGF-1 X10 (ie pTx-IGF-1X10). For example, pTx-IGF-1X10 (SEQ ID NO 16) obtained by ligation of IGF-1X10 into pTX, cleaved with Clal at 5' and Sah at 3' can be used.

In some embodiments, the pTX plasmid contains IGF-1Ec or IGF-1Ea. For example, pTx-IGF-1Ec is obtained by ligation of IGF-1Ec (SEQ ID NO 26) in pTX, and pTx-IGF-1Ea is obtained by ligation of IGF-1Ea (SEQ ID NO 24) in pTX. These plasmids express the IGF-1Ec isoform (SEQ ID NO 25) or the IGF-1 Ea isoform (SEQ ID NO 23), respectively.

IGF-1 isoforms or IGF-1 encoding plasmids described herein may include modifications from wild-type human IGF-1 isoforms. These modified sequences may include sequences with at least 80% identity, more preferably at least 90% identity, and most preferably at least 95% identity when aligning these modified sequences with wild human IGF-1 isoform sequences. type to the maximum. Methods for aligning sequences for comparison are well known in the art. In particular, the alignment algorithm disclosed in the NCBI Basic Local Alignment Search Tool (BLAST) of the National Center for Biological Information (NBCI, Bethesda, Md.) website and used in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx.

6.3.1.4. Plasmid DNA encoding SDF-1α

Stromal cell-derived factor 1 (SDF-1), also known as C-X-C chemokine 12 (CXCL12), is a chemokine protein that is encoded in humans by the CXCL12 gene on chromosome 10. It is ubiquitously expressed in many types tissues and cells. Stromal-derived factors 1a (SDF-1α) and 1p (SDF-1p) are small cytokines that belong to a family of chemokines whose members activate leukocytes and are often induced by pro-inflammatory stimuli such as lipopolysaccharide, TNF (tumor necrosis factor) or IL1 (interleukin-1). SDF-1 is produced in two forms SDF-1α/CXC1_12a and SDF-1β/CXCL12b through alternative splicing of the same gene.

Plasmids encoding one or more human SDF-1 isoforms have been designed and analyzed for the treatment of peripheral vascular disease as described in US Application No. 15/514244, incorporated herein by reference in its entirety. In particular, the polynucleotide encoding SDF-1α effectively stimulated vascular endothelial cell migration and angiogenesis when co-administered with a polynucleotide encoding human HGF.

Plasmids encoding one or more SDF-1 isoforms can be used in various embodiments of the present disclosure. In some embodiments, a plasmid encoding one or more SDF-1 is used, disclosed in US application No. 13/514244. In some embodiments, a plasmid containing the SDF-1α coding sequence is used. In some embodiments, a plasmid containing the SDF-1β coding sequence is used. In some embodiments, a plasmid encoding both SDF-1α and SDF-1β is used. In a specific embodiment, a plasmid containing the sequence of SEQ ID NO 36 is used.

6.3.2. Buffer

The liquid composition further contains a buffer to maintain the pH of the pharmaceutical composition. This buffer may include a buffer compound known in the art such as TAPS, bicine, Tris, tricine, TAPSO, HEPES, TES, MPOS, PIPES, cacodylate, or MES. This buffer may contain citric acid, monopotassium phosphate, boric acid or diethylbarbituric acid. This buffer may be a PBS, HEPES, TRIS or TRIS/EDTA buffer. This buffer may be another phosphate buffer. Phosphate buffers may contain a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate.

In particular, this buffer may be a potassium phosphate buffer. The potassium phosphate buffer may contain potassium phosphate at a concentration of 5 mM to 15 mM, 7.5 mM to 12.5 mM, 9 mM to 11 mM, or 10 mM.

The buffer contained in this liquid composition may have a pH of from 7 to 9. In some embodiments, the pH is from 7.0 to 8.5 or 8.0. In some embodiments, the pH is from 7.0 to 8.0.

In some embodiments, this liquid composition contains 7.5-12 mM potassium phosphate buffer pH up to 7 to 9. In some embodiments, this liquid composition contains 7.5-12 mM potassium phosphate buffer pH up to 7.0 to 8, 0. In some embodiments, this liquid composition contains 9-11 mm potassium phosphate buffer with a pH of up to 7.0 to 8.5. In some embodiments, this liquid composition comprises 10 mM potassium phosphate buffer pH 8.0.

6.3.3. Carbohydrate

The stability of the plasmid DNA in a given lyophilized pharmaceutical composition can be enhanced by preparing the plasmid DNA prior to lyophilization with an aqueous solution containing a stabilizing amount of carbohydrate. This carbohydrate may be mannitol or sucrose.

This carbohydrate can be added to the liquid composition so as to have a final carbohydrate concentration of from about 0.05% to about 30%, from about 0.1% to about 15%, from about 0.2% to about 15%, from about 0 .2% to about 10%, from about 0.5% to about 10%, from about 1% to about 5%, from about 1% to about 3%, from about 0.75% to 1.1%, from about 0.9% to 1.1%, from about 1.0% to about 1.1%.

In some embodiments, the liquid composition has a final concentration of at least one carbohydrate greater than 0.1% and less than 15%, greater than 0.2% and less than 10%, greater than 0.3% and less more than 7.5%, more than 0.5% and less than 5%, more than 0.5% and less than 3%, more than 0.5% and less than 2%, more than 0.5% and less than 1.1%, more than 0.75% and less than 1.1%, more than 0.9% and less than 1.1% or 1.0 %.

In particular, this liquid composition may have a final sucrose concentration of from about 0.05% to about 30%, from about 0.1% to about 15%, from about 0.2% to about 15%, from about 0.2% to about 10%, from about 0.5% to about 10%, from about 1% to about 5%, from about 1% to about 3%, from about 0.75% to about 1.1%, from about 0 .9% to about 1.1% or about 1.0%.

In some embodiments, the liquid composition has a final sucrose concentration greater than 0.1% and less than 15%, greater than 0.2% and less than 10%, greater than 0.3% and less than 7 .5%, greater than 0.5% and less than 5%, greater than 0.5% and less than 3%, greater than 0.5% and less than 2%, greater than 0 .5% and less than 1.1%, more than 0.75% and less than 1.1%, more than 0.9% and less than 1.1% or 1.0%.

In some embodiments, the liquid composition has a final mannitol concentration of less than 5%, less than 4%, less than 3%. In some embodiments, this liquid composition has a final concentration of mannitol from 0% to 10%, from 1% to 9%, from 2% to 7.5%, from 2% to 3%, from 2% to 5%, from 2% up to 4%, from 0% to 3%, from 0% to 2%, from 1% to 2%, from 1.5% to 3% or 2%.

In some embodiments, the liquid composition has a final concentration of mannitol greater than 0% and less than 10%, greater than 0% and less than 7.5%, greater than 0% and less than 5%, greater than greater than 0% and less than 4%, greater than 0% and less than 3%, or greater than 0% and less than 2.5%, or greater than 1% and less than 2.5% .

In some embodiments, the liquid composition has a final concentration of sucrose greater than 0.5% and less than 1.1% and a final concentration of mannitol greater than 0% and less than 3%. In some embodiments, this liquid composition has a final concentration of sucrose from 0.7% to 1.1% and a final concentration of mannitol from 1.5% to 3% or 2%. In some embodiments, this liquid composition has a final concentration of sucrose from 0.9% to 1.1% and a final concentration of mannitol from 1.5% to 3% or 2%. In some embodiments, this liquid composition has a final sucrose concentration of 1.0% and a final concentration of mannitol from 1.5% to 3%, from 2% to 3%, or 2%.

In some embodiments, another carbohydrate is used in the composition. This carbohydrate may be a mono-, oligo-, or polysaccharide such as sucrose, glucose, lactose, trehalose, arabinose, pentose, ribose, xylose, galactose, hexose, idose, mannose, talose, heptose, fructose, gluconic acid, sorbitol, mannitol, methyl-a-glucopyranoside, maltose, isoascorbic acid, ascorbic acid, lactone, sorbose, glucaric acid, erythrose, threose, allose, altrose, gulose, erythrulose, ribulose, xylulose, psicose, tagatose, glucuronic acid, galacturonic acid, mannuronic acid acid, glucosamine, galactosamine, neuraminic acid, arabinans, fructans, fucans, galactans, galacteronans, glucans, mannans, xylans, levan, fucoidan, carrageenan, galactocarolose, pectins, pectic acids, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, cyclodextrin, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthan gum or starch.

6.3.4. Salt

The liquid composition further comprises a salt. This salt may be NaCl or KCl.

In some embodiments, the liquid composition contains salt at a concentration greater than 0.1% and less than 0.9%, greater than 0.25% and less than 0.75%, greater than 0.4% and less than 0.6%, more than 0.4% and less than 0.5% or at a concentration of 0.45%. In some embodiments, this liquid composition contains salt at a concentration of 0.1% to 0.9%, 0.1% to 0.6%, 0.25% to 0.75%, 0.4% to 0 .6%, from 0.4% to 0.5% or at a concentration of 0.45%.

In some embodiments, this liquid composition contains NaCl at a concentration greater than 0.1% and less than 0.9%, greater than 0.25% and less than 0.75%, greater than 0.4% and less than 0.6%, more than 0.4% and less than 0.5% or at a concentration of 0.45%. In some embodiments, this liquid composition contains NaCl at a concentration of 0.1% to 0.9%, 0.1% to 0.6%, 0.25% to 0.75%, 0.4% to 0 .6%, from 0.4% to 0.5% or at a concentration of 0.45%.

In some embodiments, this liquid composition contains KCl at a concentration greater than 0.1% and less than 0.9%, greater than 0.25% and less than 0.75%, greater than 0.4% and less than 0.6%, more than 0.4% and less than 0.5% or at a concentration of 0.45%. In some embodiments, this liquid composition contains KCl at a concentration of 0.1% to 0.9%, 0.25% to 0.75%, 0.4% to 0.6%, 0.4% to 0 .5% or at a concentration of 0.45%.

In some embodiments, a different salt is used in the composition. This salt may be a monovalent cationic halide salt (e.g. sodium chloride, potassium chloride, sodium bromide, lithium chloride, sodium iodide, potassium bromide, lithium bromide, sodium fluoride, potassium fluoride, lithium fluoride and/or lithium iodide), divalent or trivalent salt (for example, calcium chloride, magnesium chloride, calcium sulfate, sodium sulfate, magnesium sulfate, chromium trichloride, chromium sulfate, sodium citrate, iron(III) chloride, yttrium(III) chloride, potassium phosphate, potassium sulfate, potassium chloride, sodium acetate, sodium phosphate, potassium phosphate, iron(II) chloride, iron(III) chloride, or a combination thereof.

6.4. Lyophilization Conditions

The lyophilized composition of the present invention is obtained by lyophilization of the liquid composition described herein. This liquid composition can be lyophilized under standard lyophilization conditions known in the art, or modifications thereof.

The method for performing lyophilization of a liquid composition of the invention may include (a) loading a container with a liquid composition at an initial temperature of from about 5°C to about -50°C; (b) cooling the DNA preparation to temperatures below zero (eg, -10°C to -50°C); and (c) essentially drying the DNA preparation. Conditions for lyophilization, such as temperature and duration, of the DNA preparation of the invention can be adjusted by one of ordinary skill in the art, taking into account factors that affect lyophilization parameters, such as the type of lyophilization unit used, the amount of DNA used, and the size of the container used.

In some embodiments, steps (b) of cooling and (c) of drying are carried out with varying temperatures. For example, the freezing step can be carried out with increasing temperatures from -50°C to -40°C, from -50°C to -30°C, from -50°C to -20°C, from -50°C to -10 °C or -50°C to 0°C. In some embodiments, the freezing step can be carried out at temperatures as low as -40°C to -50°C, -30°C to -50°C, -20°C to -50°C, -10°C to - 50°C, 0°C to -50°C or 5°C to -50°C. In some embodiments, the freezing step is carried out by decreasing the temperature and then increasing the temperature.

Some amorphous products (such as mannitol or glycine) form a metastable glass with incomplete crystallization when first frozen. These products can benefit from a heat treatment method also called annealing. During annealing, the temperature of the product changes cyclically (for example: from -40°C to -20°C for several hours and then back to -40°C; from -50°C to -20°C, holding for two (2 ) hours, then introducing a vacuum, or from -50°C to -20°C and then back to -50°C to obtain a more complete crystallization. Annealing has the additional advantage of greater crystal growth and a corresponding shorter drying time. amorphous phase can be further removed during secondary drying.

In some embodiments, the drying step is carried out in two stages - (i) primary drying and (ii) secondary drying.

In some embodiments, the primary drying is carried out while maintaining the temperature or by raising or lowering the temperature. In some embodiments, primary drying is carried out while maintaining the temperature at -50°C, -40°C, -30°C, -20°C, -10°C or 0°C. In some embodiments, the secondary drying is carried out while maintaining the temperature or by raising or lowering the temperature. In some embodiments, secondary drying may be carried out with an increase in temperature from -50°C to 20°C, from -40°C to 20°C, from -30°C to 20°C, from -20°C to 20°C, -10°C to 20°C, -50°C to 10°C, -40°C to 10°C, -30°C to 10°C, -20°C to 10°C or from -10°С to 10°С.

During the lyophilization process, phase separation may occur. For example, a pure crystalline phase may separate from a saturated amorphous phase. This crystalline phase may include ice or other crystallizing excipients. Thus, during primary drying, the pure ice phase may be removed, leaving behind other crystalline phases and any saturated amorphous phases. Conditions for primary drying can be adjusted to efficiently remove this free water while maintaining precipitate structure and DNA stability. Primary drying (sublimation) is a slow process carried out at colder temperatures, safely lower than the product's critical collapse temperature. Sublimation requires thermal energy to drive the process of phase change from solid to gaseous. When lyophilizing a product, it is necessary to consider all three methods of heat transfer - conductive heat transfer, convection and radiation.

Each liquid composition has a unique critical temperature. It is necessary to maintain the composition at a temperature safely below this critical temperature during primary drying in order to avoid degradation. This temperature depends on the vapor pressure at the ice contact surface, and in turn, this vapor pressure depends on both the rate of heat transfer to the composition (which is controlled by adjusting the shelf temperature) and the vacuum level set point of the system. During primary drying, system pressure and shelf temperature are set and controlled in combination to obtain a suitable product temperature. With the temperature and pressure settings set, the primary drying then continues for a sufficient length of time to sublimate all of the ice crystals.

In addition to the free ice that sublimates during the primary drying, there remains a significant amount of water molecules that are bound to the product. This is the water that is removed (desorbed) during secondary drying. Since all free ice has been removed during the primary drying, the temperature of the product can now be increased significantly without fear of melting or breaking. Secondary drying actually begins during the primary phase, but at elevated temperatures (typically in the range of 20 to 50°C), desorption proceeds much faster. Secondary drying rates depend on the temperature of the product. The vacuum of the system can be kept at the same level as that used during the primary drying; lower vacuum levels will not improve secondary drying time.

Secondary drying continues until the product has an acceptable moisture content for long-term storage. Depending on the application, the moisture content of fully dried products is typically between 0.5% and 3%. In most cases, the drier the product, the longer its shelf life will be. However, certain complex biological products may actually become too dry for optimal storage results, and the secondary drying method must be controlled accordingly.

The secondary drying step can be carried out with increasing temperatures from -50°C to 20°C, from -40°C to 20°C, from -30°C to 20°C, from -20°C to 20°C, from - 10°С to 20°С, from -50°С to 10°С, from -40°С to 10°С, from -30°С to 10°С, from -20°С to 10°С, from - 10°C to 10°C.

6.5. Lyophilized composition in a single dose

Another aspect of the present invention is a single dose lyophilized pharmaceutical composition. In some embodiments, the unit dosage form is a vial, ampoule, bottle, or pre-filled syringe. In some embodiments, a single dose contains from about 50 μg to 1 g of plasmid DNA, from 100 μg to 1 g of plasmid DNA, from 100 μg to 100 mg of plasmid DNA, from 1 mg to 100 mg of plasmid DNA, from 10 mg to 100 mg of plasmid DNA, 10 mg to 50 mg plasmid DNA. A single dose may contain about 10 μg, 50 μg, 100 μg, 1 mg, 10 mg, 100 mg, or 1 g of plasmid DNA. The unit dosage form may contain 0.01 mg, 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 8 mg, 10 mg, 12.5 mg, 16 mg, 24 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg or 200 mg of plasmid DNA.

In typical embodiments, the unit dosage form is a vial containing 50 mg, 10 mg, 7.5 mg, 5 mg, 1 mg, 100 μg or 50 μg of a lyophilized pharmaceutical composition suitable for administration after reconstitution. Administration includes subcutaneous, intradermal, or intramuscular administration using preloaded syringes, auto-injectors, and auto-injecting pens, each containing a predetermined amount of the pharmaceutical composition described above.

The unit dose per container may be determined based on various factors such as the active ingredient (eg, plasmid DNA), the disease being treated, the subject, route, and route of administration. A single dose may be determined based on in vitro or in vivo studies, including clinical trials.

The single dose container may be sealed and stored for an extended period of time at various temperatures (eg, room temperature to about -180°C, preferably from about 2-8°C to about -80°C, more preferably from about -20°C to about -80°C and most preferably at about -20°C).

Freeze-dried DNA compositions stored in a container are preferably stable within the range of from about 2-20°C to about -80°C for a period of at least 1 month, 3 months, 6 months or 1 year without significant loss of activity. The shelf life can be as long as several months, 1 year, 5 years, 10 years, 15 years, or up to 20 years. Preferably, the formulation is stable for a period of at least about 3 years.

6.6. Characteristics of the lyophilized composition

The present invention provides a lyophilized pharmaceutical composition having one or more desirable properties as a pharmaceutical product. These properties may include the stability and effectiveness of the active ingredient under different storage conditions, precipitation, uniform dissolution of a given formulation for administration, less contaminants, etc. The various properties provided herein can be used to select a preferred lyophilized composition or to determine ideal storage conditions for a lyophilized composition.

The stability of the plasmid DNA in a given lyophilized composition can be determined based on methods known in the art. In particular, stability can be determined based on the conformation of the plasmid DNA, for example, whether they exist in a more stable supercoiled form or a less stable open circular or linear form. The conformation of plasmid DNA can be determined by capillary electrophoresis of a sample containing plasmid DNA. The content of supercoiled DNA compared to the open circular or linear form can be measured under different conditions. For example, supercoiled DNA content can be determined before, during, or after lyophilization, or before, during, or after dissolution of a given lyophilized composition. Supercoiled DNA content can also be determined before, during, or after storage at various temperatures to select a stable lyophilized composition as well as to determine ideal storage conditions.

Another way to determine the stability of plasmid DNA is based on DNA concentrations. Various methods for measuring DNA concentrations known in the art can be used. For example, DNA concentrations can be measured based on light absorption at 260 nm. This method may further include the measurement of contaminants to better quantify the concentrations of DNA in the sample.

The appearance of the precipitate may be another important characteristic of the lyophilized product. Generally, a uniform and first class appearance of the precipitate is preferred. The non-ideal appearance of the precipitate can affect the quality of the product, for example, making it difficult to determine the quality of the product based on visual inspection, or making it difficult to extract the entire amount of the active ingredient in the container. In addition, partial or complete remelting of the precipitate may lead to instability and degradation of the active ingredient. Melting back is a type of sediment breakdown and is caused by a change from a solid to a liquid state. That is, there is incomplete sublimation (change from solid to gaseous state) in the vial. These changes may include a change in the physical form of the drug substance and a pocket of moisture.

The appearance of the precipitate may be determined by visual inspection, which may include photography. Visual checks can be carried out mainly on the basis of historical precedent. Before judging the quality of a product, a reliable quality evaluation program for visual inspection is essential. This qualitative assessment program may be based on past experience or published information. Specific visual inspection guidelines may be developed for the medicinal product, for example, based on the information provided in the inspection guide "Lyophilization of Parenterals: Guide To Inspections of Lyophilization of Parenterals (7/93)" published by the US Food and Drug Administration and medicines.

Another characteristic of the lyophilized product that can affect the quality of the drug product is the turbidity of the dissolved drug obtained by dissolving the lyophilized product. The turbidity of the dissolved drug may correlate with the release of the active ingredients in a given lyophilized composition. In general, complete dissolution is preferred, i.e. low turbidity of the isolated drug. Incomplete dissolution can result in waste of the active ingredients of the pharmaceutical composition and blockage of the syringe for administering the dissolved drug.

The turbidity of the dissolved drug can be measured by visual inspections or by measuring light absorption at certain wavelengths, for example at 450 nm and 650 nm. Light absorption can be measured using a device available in the art, such as the ThermoMAX microplate from Molecular Devices.

Dissolution time is another factor that can be related to the quality of the drug product. In general, a short dissolution time is preferred. Extended dissolution time at the user step may result in a partial loss of efficacy if the drug is not completely dissolved, as it is common to use infusion filters during administration to a patient. The time required to dissolve the lyophilized product can be determined, for example, by measuring the turbidity of the dissolved drug at different time points after dissolution. For example, turbidity can be measured after 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2 hours or more.

Residual moisture after freeze-drying can also be important. Various methods known in the art can be used to measure residual moisture in a lyophilized composition. For example, a Karl Fisher Coulometer C20 (Mettler Toledo) can be used to analyze moisture content. A pocket of moisture in a lyophilized composition can lead to greater instability and increased degradation of the product. Thus, a lower moisture content may be preferred.

6.7. Methods for treating a disease using a lyophilized composition

Methods for treating diseases using a lyophilized composition are within the scope of the present invention.

6.7.1. Dissolution of the lyophilized composition

Various dissolution methods can be used, such as vortex mixing methods, mechanical orbital shaker methods, or stationary vial methods.

The final concentration of the dissolved drug for administration may be adjusted depending on many factors, including the amount of drug to be delivered, the age and weight of the subject, the method of delivery, the route and immunogenicity of the delivered antigen.

The lyophilized composition of the present invention can be dissolved using a suitable solution such as water, TE, PBS (phosphate buffered saline), Tris buffer or normal saline to a final concentration of about 10 mg/ml, 5 mg/ml, 1 mg /ml, 0.5 mg/ml, 0.3 mg/ml, 0.2 mg/ml, 0.1 mg/ml or 0.05 mg/ml.

6.7.2. Administration of dissolved drug

The dissolved drug of the present invention can be administered to a mammalian subject for the treatment of various diseases. The dissolved lyophilized composition of the invention can be administered by various delivery routes, for example, orally or via parenteral routes such as intravenous, intramuscular, intraendocardial, intramyocardial, intrapericardial, intraventricular, intraarticular, intradermal, intracerebral, intrarenal, intrahepatic, intrasplenic, intralymphatic, subcutaneous, intraperitoneal , intratesticular, intraovarian, intrauterine, sternal, intratracheal, intrapleural, intrathoracic, intradural, intraspinal, intramedullary, intramural, intrascorionic, and arterial injection or infusion, or topically via rectal, intranasal, inhalation, or intraocular administration. In some embodiments, the delivery method is intramuscular, intramyocardial, intravenous, intracerebral, or intrarenal delivery.

In typical embodiments, the nucleic acid construct is administered by injection of a liquid pharmaceutical composition. In presently preferred embodiments, the polynucleotide construct is administered by intramuscular injection. Typically, this polynucleotide construct is administered by intramuscular injection close to the affected area. In some embodiments, the polynucleotide constructs are introduced into the muscles of the limbs, the heart, or other parts of the subject's body.

In some embodiments, the construct is injected subcutaneously or intradermally. In some embodiments, the polynucleotide construct is administered via intravascular delivery. In some embodiments, the construct is injected by retrograde intravenous injection.

It should be understood that the typical daily dose of the dissolved lyophilized composition of the present invention should be determined in light of various relevant factors including the conditions to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, the severity of the symptom of the patient, and it can be administered in single dose or in divided doses. The polynucleotide construct is administered at a therapeutically effective dose.

In some embodiments of the methods described herein, the polynucleotide construct is administered at a total dose of 1 μg to 200 mg, 1 mg to 200 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 20 mg, 5 mg to 10 mg, 16 mg, 8 mg or 4 mg.

In typical embodiments, the total dose is divided into a number of individual injection doses. In some embodiments, the total dose is divided into a number of equal injection doses. In some embodiments, the total dose is divided into unequal injection doses.

In various divided dose embodiments, the total dose is administered at 4, 8, 16, 24, or 32 different injection sites.

In some embodiments, the injection dose is 0.1-5 mg. In certain embodiments, the injection dose is 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, or 0.5 mg .

This total dose may be administered at one visit or at two or more visits.

In exemplary split dose embodiments, all of the range of injection doses are administered within 1 hour of each other. In some embodiments, all of the range of injection doses are administered within the range of 1.5; 2; 2.5 or 3 hours apart.

In various embodiments of these methods, the total dose of the polynucleotide construct, whether administered as a single unitary dose or divided into multiple injection doses, is administered to the subject only once.

In some embodiments, one cycle may include administering a total dose of the polynucleotide construct to multiple injection sites in one, two, three, or four visits. In particular, one cycle may include the administration of 32 mg, 16 mg, 8 mg, or 4 mg of the polynucleotide construct at multiple injection sites over two visits. These two visits can be separated from each other by 3, 5, 7, 14, 21 or 28 days.

In some embodiments, the cycle may be repeated. This cycle can be repeated twice, three times, four times, five times, six times or more.

In some embodiments, this cycle can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months after the previous cycle.

In some embodiments, the total dose administered in the subsequent cycle is the same as the total dose administered in the previous cycle. In some embodiments, the total dose administered in the subsequent cycle is different from the total dose administered in the previous cycle.

In presently preferred embodiments, the nucleic acid construct is administered at a dose of 8 mg per affected limb, evenly divided into a number of intramuscular injections and a number of visits, where each of the number of injections in any one visit is given to a separate injection site. In some embodiments, the nucleic acid construct is administered at a dose of 8 mg per affected limb evenly divided into a first dose of 4 mg per limb on day 0 and a second dose of 4 mg per limb on day 14, where each of the first and second doses is evenly divided for multiple injection doses.

The actual amount administered, the rate and dynamics of administration will depend on the nature and severity of the disease being treated. In exemplary embodiments, the polynucleotide construct is administered in an effective amount to reduce symptoms of a disease, such as pain. In some embodiments, this amount is effective to reduce the symptom within 1 week after administration. In some embodiments, the amount is effective to reduce a symptom within 2 weeks, 3 weeks, or 4 weeks of administration.

This plasmid DNA may be administered alone or in combination with other plasmid DNA, either simultaneously or sequentially, depending on the condition being treated.

In some embodiments, the dissolved composition contains plasmid DNA encoding human HGF. This dissolved drug can be administered to treat a variety of diseases, for example a disease that has previously been shown to be treatable by the introduction of plasmid DNA. This plasmid DNA may encode a therapeutic gene, such as human HGF. This disease includes ischemic or hepatic disease, coronary artery disease ("CAD"), amyotrophic lateral sclerosis ("ALS"), peripheral arterial disease ("diabetic ulcer"), and diabetic peripheral neuropathy ("DPN") or neuropathy caused by diseases injuries, infections or vitamin deficiency conditions, but is not limited to them. For example, neuropathy may be caused by diabetes, vitamin deficiencies, autoimmune diseases, genetic or hereditary disorders, amyloidosis, uremia, toxins or poisons, trauma or injury, tumors, or may be idiopathic. The methods described in US Pat. 7838505; 7745174; 8338385; 8389492 and US applications No. 12/359137; 14/355792; 15/030999. References herein are incorporated herein by reference in their entirety.

In some embodiments, the dissolved composition contains plasmid DNA encoding human IGF-1. This dissolved drug may be administered to treat a variety of diseases, for example a disease that has previously been shown to be treatable by administration of plasmid DNA encoding human IGF-1 or human IGF-1 protein. This disease includes, but is not limited to, neuropathy caused by diseases, injuries, infections, or vitamin deficiency conditions. For example, this neuropathy may be caused by diabetes, vitamin deficiencies, autoimmune diseases, genetic or hereditary disorders, amyloidosis, uremia, toxins or poisons, trauma or injury, tumors, or may be idiopathic. In some embodiments, a plasmid encoding human IGF-1 protein (pTx-IGF-1) is administered with another plasmid encoding human HGF (eg, VM202) to treat a disease. For the treatment of diseases by administering a lyophilized composition, the methods described in US applications No. 16/513560 and/or 16/513564 can be used.

In some embodiments, the dissolved composition contains plasmid DNA encoding human SDF-1α. For the treatment of a disease, the plasmid can be administered alone or together with other plasmid DNA. In some embodiments, a plasmid encoding human SDF-1α (eg, pCK-SDF-1α) is introduced with another plasmid DNA encoding human HGF (eg, VM202) to treat a disease. This disease includes, but is not limited to, a vascular disease such as peripheral vascular disease. The methods described in US application No. 15/514244 can be used to treat a disease by introducing a plasmid encoding human SDF-1α.

6.8. Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a full disclosure and description of how to use the present invention, and are intended neither to limit the scope of what the present inventors consider to be their invention nor to represent that that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg amounts, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations can be used, for example, p.o. - pair(s) of bases; kbp - thousand (chi) pairs of nucleotides; pl - picoliter(s); s - second(s); min - minute (you); h - hour(s); ak - amino acid (you); nt - nucleotide(s) and the like.

In the practice of the present invention, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, genetic engineering, and pharmacology will be used that are within the skill of the art.

6.8.1. Example 1: Lyophilized composition VM202 (Study 001) 6.8.1.1. Analyzed pharmaceutical composition containing VM202

Received different preparations containing VM202, as shown below in TABLE 1. The active ingredient - VM202 plasmid - was obtained from frozen stock solutions containing either 1.6 mg/ml VM202 in 0.9% NaCl, or 1.3 mg/ml VM202 in 0.9% NaCl.

These formulations were prepared using commercially available materials and equipment, e.g., potassium phosphate monobasic (Spectrum, cat. no. PO200), potassium phosphate dibasic (EMD, cat. no. PX1570-1), mannitol (J.T. Baker, cat. no. 2553-05), sucrose (J.T. Baker, cat. no. 4074-05), sodium chloride (Millipore, cat. no. 1.06404.5000) and trehalose (Pfanstiehl, cat. no. T-104-04), dialysis cassette (Thermo Scientific, cat. no. 66380 (10000 MWCO molecular weight cut-off)), glass vials (3 ml or 20 ml glass vials (Schott, Type I, borosilicate, cat. no. 68000316 or 68000321)) and stoppers (13 mm or 20 mm single vent lyocrap, Flurotec®, ( West Pharmaceutical, cat. no. 19700034 or 19700311)).Excipients such as dextran (MP Biomedicals, cat. no. 101514), lithium hydroxide (Sigma Aldrich, cat. no. L4533-100G) and phosphoric acid (J.T. Baker, cat. no. 0262- 01).

6.8.1.2. Analysis of different drugs on a small scale

6.8.1.2.1. Sample preparation

To obtain different preparations, vials with VM202 at a concentration of 1.6 mg/ml in 0.9% NaCl were removed from storage at -70°C and thawed at ambient temperature.

Small scale 1 round samples: KP8M2SN (see Table 1) was then prepared by dialysis using a Thermo dialysis cassette with MWCO 10,000 at 10,000 or more volume changes relative to the target buffer preparation (10 mM potassium phosphate, 2% mannitol, 1.0% sucrose, 0.1% NaCl, pH 8.0) for 24 hours at 2-8°C. After dialysis, the concentration of KP8M2SN was adjusted to 0.5 mg/ml VM202 using preparation buffer.

KP8MS3N, KP8MT3N and control were obtained by diluting the drug. Specifically, 1.7 ml of VM202 (i.e. VM202 in 0.9% NaCl) was diluted with 0.85 ml of dilution buffers (buffer #2 for KP8MS3N, buffer #3 for KP8MT3N, and buffer #8 for control) given in TABLE 2. After dilutions, each preparation was adjusted to contain 0.5 mg/ml VM202 for KP8MS3N and KP8MT3N, respectively, using preparation buffer containing 0.45% NaCl.

Samples for the 2nd round of small scale assays: 4MSN, 3MSN and 2MSN were prepared by diluting 0.84 ml drug substance (i.e. VM202 in 0.9% NaCl) using 0.42 ml dilution buffers (buffer 4 for 4MSN, 3MSN Buffer 5, and 2MSN Buffer 6), also listed below in TABLE 2. After dilution, each preparation, except for the control, was adjusted to include 0.5 mg/ml VM202 using each preparation buffer containing 0.45% NaCl.

Preparation of samples for stability studies with accelerated degradation: for studies of stability with accelerated degradation, a separate set of preparations was additionally received. For this single kit, 4MSN, 3MSN, 2MSN, 2M1SN and control were prepared by diluting the drug substance (i.e. VM202 in 0.9% NaCl) with the dilution buffers listed above in TABLE 2 in a 2:1 ratio (buffer 4 for 4MSN , buffer 5 for 3MSN, buffer 6 for 2MSN, buffer 7 for 2M1SN, and buffer 8 for control). After dilutions, all preparations, except for the control, were adjusted to contain VM202 at a concentration of 0.5 mg/ml using each preparation buffer containing 0.45% NaCl.

A simulant was prepared using 0.5 mg/mL dextran in 0.9% NaCl and 1.1% sucrose.

Lyophilization of samples: after adjusting the concentration to 0.5 mg/mL, formulated VM202 was sterilized filtered through a 0.2 µm PES filter (small scale and accelerated degradation studies) or cellulose acetate (CA) filter ( additional stability study with accelerated degradation) under aseptic conditions. All samples were aseptically filled into sterilized 3 ml vials with a fill volume of 0.75 ml per vial for small scale lyophilization or sterilized 20 ml vials with a fill volume of 5 ml per vial for accelerated degradation stability studies in a microbiological safety cabinet. Specifically, filling with these drugs was done using a syringe filter (PALL Life Science, 13 mm Acrodisc with 0.2 µm PES membrane, catalog # 4602) using a vacuum filtration system (Corning, 1 L vacuum filter/storage bottle system, 0 .2 µm CA membrane, P/N 431-205; Thermo scientific, fast current filter element, 0.2 µm PES membrane, P/N 567-0020) in a microbiological safety cabinet (NuAire Microbiological Safety Cabinet, Class II, Type A/ B3, Model No. NU-425-600)).

After filling, these vials were partially stoppered with sterile caps and loaded into a lyophilizer (VirTis (Model No. 25L Genesis SQ Super XL-70) for lyophilization using the cycle described in TABLE 3. The mock vials were used to completely surround the VM202 vials such that during drying process for lyophilization on a small scale provides a comparable radiant heat transfer from adjacent vials.

After lyophilization, these vials were completely stoppered under a partial vacuum of 600 mTorr (79.99 Pa), crimped with aluminum caps and labeled. Labels included project information, date, incubation temperature/stress condition, and point in time. After labeling, the samples were placed under appropriate stability conditions.

6.8.1.2.2. Analyzes for testing different drugs

Lyophilized preparations were subjected to various stress conditions to assess their relative stability. For thermal stress exposure of the samples, all preparations were stored at 5°C, 25°C or 40°C for up to 10 weeks. To assess post-dissolution stability, samples were dissolved in a biosafety cabinet using a target volume of 5.0 ml filtered water for injection (WFI), recapped, and resealed. After dissolution, the samples were stored at 25°C for 3 or 7 days. The different conditions analyzed in this experiment are summarized below in TABLE 4.

The following analyzes were performed to analyze the stability of different drugs under different conditions.

(1) Visual inspection: A visual inspection was carried out against a dark and white background. We received digital photographs.

(2) UV (ultraviolet) spectrophotometry: UV spectrophotometry analysis was performed using an Implen nanophotometer with a cap factor of 10. The plasmid DNA concentration of the test samples was determined by absorbance measurement at 230, 260, 280, and 350 nm. Concentration analyzes were performed using the ViroMed UV/Vis protocol. The calculations were carried out using the following equations:

Concentration (µg/ml) = [(D-E)/C] × (V/A)

Yield = (low µg/mL) / (high µg/mL) × 100%

OD ^260nm / OD ^280nm =D/F

OD ^260nm / OD ^230nm =D/G

Where:

A is the mass of the sample taken to obtain the dilution.

B is the total weight of the sample and dilution buffer.

C is the extinction coefficient of a 0.005% solution in a cuvette with a path length of 1 cm (0.02).

D is the optical density for the maximum at 260 nm.

E is the absorbance measured at 350 nm.

F is the absorbance measured at 280 nm.

G is the absorbance measured at 230 nm.

Acceptance Criteria:

D should fall within the range of 0.5 to 1.5 OD units (optical density)

Output must be greater than or equal to 98%

OD ^260nm / OD ^280nm must be between 1.8 and 2.0

OD ^260nm / OD ^230nm must be greater than or equal to 1.1

(3) Capillary electrophoresis: For structural analysis of plasmid DNA (purity and supercoiled DNA), a Beckman Coulter, ProteomeLab™ PA 800 CE instrument equipped with a P/ACE™ MDQ UV detector was used. The results were analyzed using the 32 Karat program (version 7.0). Prior to analysis by capillary electrophoresis, a 40 cm Beckman Coulter Neutral coated capillary with 50 µm ID (P/N 477441) and 8 µm aperture was conditioned by passing ultrapure HPLC (high performance liquid chromatography) water at 137.9 kPa for 1 minute, followed by washing with 100 mM phosphoric acid (pH 2.6) at 137.9 kPa for 1 minute. Separation took place by applying a constant voltage of 17 kV for 14 minutes. 40 μl of the sample at a concentration of 0.5 mg/ml was added to a polyethylene insert for analysis. We used the following parameters:

• Injection pressure/time: 3.45 kPa/11 s.

• Separation buffer/pressure: 100 mM phosphoric acid (pH 2.60), 85% / 137.9 kPa; 60 s.

• Wash Buffer/Pressure: HPLC grade ultrapure water / 137.9 kPa; 60 s

• Detector wavelength: 254 nm.

• Separation voltage: 17 kV; 0.17 min; 14 min.

A typical VM202 capillary electrophoresis result is shown in FIG. 1.

(4) Haze: Haze was determined by measuring the absorbance of the sample at 450 nm and 650 nm using a ThermoMAX microplate reader from Molecular Devices.

(5) Near ambient DSC (differential scanning calorimetry) analysis: Approximately 10 μl of each preparation was frozen at -60°C using Pyris Diamond DSC with Intercooler II. At a rate of change of 5°C/min, the sample was heated to 25°C and the heat flux was recorded during the heating process. A program including an annealing step was also carried out. The annealing step was carried out by heating the sample to -15° C. at a change rate of 5° C./min.

(6) Karl Fischer Moisture Analysis: Karl Fisher Coulometer C20 (Mettler Toledo) was used for moisture analysis. AquaStar Water Standard Oven 1% was used to determine system accuracy. Before removing the assay caps, the sample vials were brought to room temperature. The weighing boats were weighed before and after adding the samples to determine the amount of lyophilized powder used for analysis. Approximately 10-160 mg of the substance was used for analysis.

6.8.1.2.3. Analysis of KP8M2SN, KP8MS3N and KP8MT3N (1st round of small scale analysis)

DSC analysis under conditions close to ambient conditions: before the lyophilization process, 10 µl of samples prepared in the form of a preparation of VM202 - i.e. KP8M2SN, KP8MS3N, KP8MT3N and control (TABLE 1) at 0.5 mg/mL were analyzed by differential scanning calorimetry (DSC) under near ambient conditions to characterize candidate drugs. Signals analyzed include fracture temperature (Tg'), devitrification temperature (Td) and the benefits of annealing different preparations with VM202 in frozen states. On FIG. 2A-2D illustrate liquid state DSC results for 1st round small scale lyophilization of KP8M2SN (Fig. 2A), KP8MS3N (Fig. 2B), KP8MT3N (Fig. 2C) and control (Fig. 2D) without annealing (left) or annealed (right) VM202. The glass transition temperature (Tg') of KP8M2SN was observed at -44°C. The control showed a eutectic melting point (Te) at about -21°C. The devitrification temperature (Td) was observed in all preparations and disappeared after annealing. Because some of the additives showed decleavage signals that disappeared after annealing, lyophilization cycle 1 (TABLE 3) was designed to include an annealing step during freezing for the 1st round on a small scale.

Increasing the freezing temperature from -50°C to -20°C and holding for two (2) hours, then the subsequent introduction of vacuum provided annealing and induction of crystallization of mannitol. Primary drying was carried out at a shelf temperature of -20° C. (24 hours) with a chamber pressure of 80 mTorr (10.66 Pa) (TABLE 3). Secondary drying at a shelf temperature of 20° C. was designed to remove residual water that did not sublimate during the primary drying step.

On FIG. 3 is a plot of temperatures (left ordinate) and pressures (right ordinate) during the lyophilization process for the 1st round of small scale analysis. The temperature of the product dropped below -36° C. due to the loss of sublimation heat when the vacuum was applied. The Pirani pressure gauge value merged with the capacitance gauge reading at approximately 30 hours during the primary drying step, which confirmed the completion of the primary drying process. Secondary drying was initiated and the samples were allowed to dry for another 13 hours. The entire cycle lasted 46 hours with a potential reduction to 43 hours if secondary drying was initiated immediately upon completion of primary drying.

At the completion of the 1st round of the small scale lyophilization cycle, all vials were sealed with a vacuum pressure of 600 torr. These lyophilizates were removed from the chamber and analyzed. The lyophilized pellet of KP8M2SN had a first class appearance. Other sediments showed signs of destruction, especially control. Dissolved samples were clear and comparable to pre-lyophilization samples (FIGS. 4A-C). The moisture content of these precipitations was 1.37% (KP8M2SN), 2.40% (KP8MS3N), 1.44% (KP8MT3N) and 1.25% (control), respectively.

The concentration of VM202 in each sample was measured after dissolution of the sample from the 1st round of the lyophilization cycle on a small scale, and the results are summarized in TABLE 5. These results showed that each sample contained VM202 at a similar concentration.

Capillary Electrophoresis: Dissolved samples were additionally checked by CE for product purity. Chromatograms and peak area data (%) after CE are shown in FIG. 5 and in TABLE 6. The results from the CE analyzes showed a decrease in the supercoiled peak area of KP8M2SN, KP8MT3N and control after lyophilization and dissolution. KP8MS3N had the largest supercoiled peak area after lyophilization and dissolution.

In conclusion, the results show that KP8M2SN containing 1.0% sucrose and 0.1% NaCl produces a better lyophilized pellet than KP8MS3N containing 0.5% sucrose and 0.45% NaCl or KP8MT3N containing 0. 5% trehalose and 0.45% NaCl. These results further indicated that KP8MS3N containing 0.5% sucrose and 0.45% NaCl had the least degradation in the CE measurement. Overall, this indicates that the sucrose-containing formulations (KP8M2SN and KP8MS3N) have better properties (eg, first-class precipitate formation and stability) than the trehalose-containing formulation (KP8MT3N).

6.8.1.2.4. Analysis of 1st set of 4MSN, 3MSN and 2MSN (2nd round of small scale lyophilization cycle)

Based on the results from the 1st round of the small scale lyophilization cycle, the next lyophilization cycle (the 2nd round of the small scale analysis) focuses on improving the appearance of the precipitate by changing the concentration of mannitol in KP8MS3N while maintaining its sucrose concentrations (0.5%) and NaCl (0.45%).

Prior to the freeze-drying process, 10 µl samples of pre-formulated VM202 - i.e. 4MSN, 3MSN, 2MSN and control (TABLE 1) - at a concentration of 0.5 mg/ml were analyzed by DSC under conditions close to environmental conditions to characterize candidate drugs. On FIG. 6A-D illustrate liquid state DSC results from the 2nd round small scale lyophilization assay. The control (Fig. 6D) showed a eutectic melting point (Te) of the order of -21°C. The devitrification temperature (Td) was observed in all preparations (no annealing, left), and it disappeared after annealing (right). Because of the observation of devitrification signals, lyophilization run 2 (TABLE 3) was designed to include an annealing step during freeze for the 2nd round of small scale analysis.

Increasing the freezing temperature from -50°C to -20°C and holding for two (2) hours, then the subsequent introduction of vacuum provided annealing and induction of mannitol crystallization. Primary drying was carried out at a shelf temperature of -20° C. (25 hours) with a chamber pressure of 50 mTorr (6.67 Pa) (TABLE 3). Secondary drying at a shelf temperature of 20° C. was designed to remove residual water that did not sublimate during the primary drying step.

On FIG. 7 shows a plot of temperature (left ordinate) and pressure (right ordinate) during the lyophilization process for the 2nd assay run on a small scale. The temperature of the product dropped below -37° C. due to sublimation heat loss when the vacuum was applied. The Pirani pressure gauge value merged with the capacitance gauge reading at approximately 11 p.m. during the primary drying step, confirming the completion of the primary drying process. Secondary drying was initiated and the samples were allowed to dry for another 13 hours. The entire cycle lasted 47 hours with a potential reduction to 42 hours if secondary drying was initiated immediately upon completion of primary drying.

Once the cycle was completed, the samples were removed from the lyophilizer and analyzed. By visual inspection, 4MSN showed first-class sediment. 3MSN and 2MSN also showed a good dry-out pellet, while the control showed signs of pellet breakdown after lyophilization. The samples were clear and colorless after dissolution, comparable to the samples before lyophilization (Fig. 8A-C). The moisture content of the sediment was 2.51% (4MSN), 2.15% (3MSN), 2.01% (2MSN) and 1.12% (control).

The concentration of VM202 in each sample was measured after dissolving the sample from the 2nd round of the lyophilization cycle on a small scale, and the results are summarized in TABLE 7. These results showed that each sample contained VM202 at a similar concentration.

Capillary electrophoresis: dissolved samples were further checked for product purity by CE. Chromatograms and results in tabular form from CE are detailed in FIG. 9 and TABLE 8. 4MSN and 3MSN showed minimal degradation in supercoiled peak area, while 2MSN and control showed reductions in the supercoiled peak after lyophilization and dissolution.

These results indicated that 4MSN (10 mM potassium phosphate, 4% mannitol, 0.5% sucrose, 0.45% NaCl at pH 8.0) gave the best lyophilized pellet and the least degradation as determined by CE in 2- th round of the lyophilization cycle on a small scale.

6.8.1.2.5. Analysis of the 2nd set of 4MSN, 3MSN and 2MSN (2nd round of small scale lyophilization cycle)

Another set of 4MSN, 3MSN, 2MSN and control was made through the 2nd round of a small scale lyophilization cycle designed with a primary drying shelf temperature of -20°C at a chamber pressure of 50 mTorr (6.67 Pa) and a secondary drying shelf temperature of 20°C at a chamber pressure of 50 mTorr (6.67 Pa) (TABLE 3). On FIG. 10 shows a graph of the dynamics of temperatures and pressures obtained during this cycle. The temperature of the product dropped below -37° C. due to sublimation heat loss when the vacuum was applied. The Pirani pressure gauge values merged with the capacitive pressure gauge reading at approximately 75 hours during primary drying. Secondary drying was initiated and the samples were allowed to dry for another 13 hours. The entire cycle lasted 87 hours.

Once the cycle was completed, the samples were removed from the lyophilizer and analyzed. By visual inspection, 4MSN showed first-class sediment. 3MSN and 2MSN also showed a good dry-out pellet, while the control showed signs of pellet breakdown after lyophilization. Upon dissolution, some vials in all preparations, except for the control, showed turbidity, while all samples were clear before lyophilization (Fig. 11A-F). The moisture content of the sediment was 0.97% (4MSN), 0.95% (3MSN), 1.69% (2MSN) and 1.06% (control).

Turbidity: The concentration and turbidity results are summarized in TABLE 9. All samples showed similar concentration values after dissolution. For all preparations, no turbidity was detected before lyophilization and after dissolution.

Capillary electrophoresis: dissolved samples were further checked for product purity by CE. Chromatograms and peak percentages from CE are shown in FIG. 12 and TABLE 10. 4MSN, 3MSN and control showed a significant decrease in the supercoiled peak area after lyophilization and dissolution. 2MSN showed minimal degradation in the supercoiled peak area after lyophilization and dissolution.

These results indicated that 2MSN (10 mM potassium phosphate, 2% mannitol, 0.5% sucrose, 0.45% NaCl pH 8.0) gave the least degradation determined by CE in this separate set of experiments for 2 -th round of analysis on a small scale.

Samples from this lyophilization run were used for 10 weeks of accelerated degradation stability studies.

6.8.1.2.6. Analysis of 2MSN and 2M1SN

Whereas KP8MS3N and 2MSN containing 2% mannitol, 0.5% sucrose and 0.45% NaCl showed the least degradation as measured by CE in the 1st round assays and certain 2nd round assays in the small scale, the KP8MS3N and 2MSN preparations were further optimized by varying the sucrose concentration while keeping the concentrations of mannitol (2%) and NaCl (0.45%) at the same level. Specifically, a 2M1SN preparation containing 2% mannitol, 0.45% NaCl and 1.0% sucrose was prepared and analyzed in comparison to 2MSN.

2M1SN and 2MSN were lyophilized using similar lyophilization cycle parameters relative to those used in the accelerated degradation stability study. On FIG. 13 shows the temperature and pressure measurements from this run. The temperature of the product dropped below -41° C. due to sublimation heat loss when the vacuum was applied. The Pirani pressure gauge values merged with the capacitance gauge reading at approximately 60 hours during primary drying. Secondary drying was initiated and the samples were allowed to dry for another 13 hours. The entire cycle lasted 76 hours with the potential to be reduced to 70 hours if secondary drying was initiated immediately upon completion of primary drying.

After lyophilization, 2M1SN showed a first-class pellet, and 2MSN showed a good pellet with little drying (Fig. 14). When dissolved, 2M1SN gave a clear solution, while 2MSN showed haze. Both formulations were clear prior to lyophilization. The moisture content of the precipitation was 1.31% (2MSN) and 1.44% (2M1SN).

Haze: The formulations were evaluated for concentration and haze and the results are summarized in TABLE 11. All samples showed similar concentration values after dissolution. For a further stability study with accelerated degradation, it was determined that turbidity measurements at 450 nm were better able to assess drug differences. For 2M1SN, no turbidity was detected before lyophilization and after dissolution. After dissolution of 2MSN, a significant increase in haze was observed at 450 nm.

Capillary electrophoresis: dissolved samples were further checked for product purity by CE. Chromatograms and results in tabular form from CE are detailed in FIG. 15 and in TABLE 12. 2MSN showed a decrease in supercoiled peak area after lyophilization and dissolution. 2M1SN showed minimal degradation in the supercoiled peak area after lyophilization and dissolution.

Samples produced in this lyophilization cycle were used for 10 weeks for further stability studies with accelerated degradation.

6.8.1.3. Stability study with accelerated degradation of 4MSN, 3MSN, 2MSN and controls

The stability of lyophilized preparations of VM202 - 4MSN, 3MSN, 2MSN and control - was evaluated under different storage conditions at refrigeration (5 plus/minus 3°C), ambient (25 plus/minus 3°C) and accelerated degradation (40 plus/minus 3°C), and dissolved preparations VM202 - at ambient temperature for 3 or 7 days.

Lyophilized samples from the accelerated degradation stability study were dissolved in 5 ml of filtered water in a microbiological safety cabinet, resealed, sealed and incubated at 25° C. for 3 and 7 days. After 3 and 7 days, the incubated samples were removed from the incubator and analyzed visually, for concentration and by CE analysis.

Visual inspection: after storage of preparations 4MSN, 3MSN, 2MSN and control at 25°C for 3 days (Fig. 16A) and 7 days (Fig. 16B), no change in appearance after dissolution was observed compared to T equal to 0.

At each time point, the samples were analyzed for absorbance at 260 nm to determine the concentration of VM202. The concentration of each drug after storage at 25°C for 3 and 7 days is similar to the results obtained in T equal to 0 (TABLE 13). All samples were within 5% of the target concentration. Therefore, temperature stress did not induce large changes in VM202 concentration.

Capillary electrophoresis: CE analysis was used to monitor the purity of the product during storage at a given temperature. The CE chromatograms and results in tabular form for preparations after storage are detailed in FIG. 17A-B and in TABLE 14. All formulations (ie 4MSN, 3MSN, 2MSN and control) showed reductions in supercoiled peak area after storage at 25°C for 3 to 7 days. 2MSN showed the best stability from 3 to 7 days with only 1.1% reduction in supercoiled peak area.

Lyophilized preparations of VM202 were stored for 10 weeks at 5°C, 25°C and 40°C (TABLE 4). At each time point, samples were removed from storage at a given temperature and evaluated visually. Photographs of the vials were taken and samples were evaluated for pellet shape. All lyophilized pellets remained intact and did not show any signs of change during storage, regardless of temperature conditions (the control pellet remained elevated). These samples were then dissolved in 5 ml filtered water and evaluated for clarity and precipitation. Most of the samples for 4MSN, 3MSN and 2MSN showed little difference in transparency compared to T=0. 18B) for 10 weeks is shown in FIG. 18A-C.

Concentration, turbidity, moisture content, and capillary electrophoresis analyzes were also performed to assess the stability of lyophilized VM202 during temperature stresses.

At each time point, the samples were dissolved and analyzed for absorbance at 260 nm to determine the concentration of VM202 in the vials. TABLE 15 shows that the concentration of VM202 in lyophilized samples after 10 weeks of storage at a given temperature is similar to the results obtained at T equal to 0. Therefore, temperature stress did not induce large changes in the concentration of VM202.

Turbidity: The turbidity of each sample was also assessed at each time point in this study. The results are detailed in TABLE 16. The turbidity of the dissolved 4MSN, 3MSN and 2MSN preparations did not change significantly after 10 weeks of storage, regardless of temperature conditions.

Moisture content was also analyzed at each time point. TABLE 17 describes the results for moisture content after 10 weeks of storage at a given temperature. After 10 weeks of storage at 5° C., 25° C. and 40° C., slight decreases in moisture content were observed in a temperature dependent manner. Higher storage temperatures resulted in lower moisture content. 2MSN had the highest moisture content during the accelerated degradation stability study.

Capillary Electrophoresis: Capillary electrophoresis analysis was used to monitor the purity of the product. CE chromatograms and peak area values after 10 weeks of incubation at 40°C are shown in FIG. 19 and in TABLE 18, respectively. All formulations showed significant reductions in supercoiled peak area with corresponding increases in open ring peak area compared to T=0 after dissolution after 10 weeks storage at 40°C. 2MSN showed the largest supercoiled peak area, although this value was significantly lower at 10 weeks of storage at 40°C than at zero. Large levels of open ring peak area were observed in preparations containing 3% and 4% mannitol, as well as in the control preparation.

Trends in CE-determined supercoiled peak purity, weighted by standard deviation and including deviation error bars, over 10 weeks at different temperatures are detailed in FIG. 20A-C - 5°C (Fig. 20A), 25°C (Fig. 20B) or 40°C (Fig. 20C). Significant differences in purity were observed after incubation at elevated temperature. As illustrated in FIG. 20A-C, 2MSN containing 2% mannitol and 0.45% NaCl showed the highest level of purity after storage at 40°C.

Trends in open ring impurity peaks detected by CE after storage at different temperatures are also shown in FIG. 21A-C. Small increases in open ring peak areas were observed at 5°C. However, at 25° C. or higher, all preparations showed significant degradation. After incubation at 40°C, higher levels of open ring impurities were observed for 4MSN, 3MSN and control. Compared to this, 2MSN showed significantly less degradation at all temperatures.

6.8.1.4. Stability study with accelerated degradation of 2MSN and 2M1SN

The stability of lyophilized preparations VM202, 2MSN and 2M1SN was also evaluated after incubation at ambient temperature (25°C) for 3 or 7 days. Samples were dissolved in 5 ml of filtered water in a microbiological safety box, resealed, sealed and incubated at 25°C for 3 and 7 days. After 3 and 7 days, the incubated samples were removed from the incubator and analyzed by visual inspection, concentration analysis and CE analysis.

Visual inspection: Visual inspection was performed at both time points (FIGS. 22A-D). Haze was observed in 2MSN after 3 and 7 days of storage at 25° after dissolution. 2M1SN remained transparent at both times.

At each time point, the samples were analyzed for absorbance at 260 nm to determine the concentration of VM202. VM202 concentrations in both formulations were targeted at T=0 and after incubation at 25° C. for days 3 and 7 after dissolution (TABLE 19). Therefore, temperature stress did not induce significant changes in VM202 concentration.

Capillary electrophoresis: Samples were also analyzed for product purity by CE after storage at a given temperature. The CE chromatograms and peak area results of the samples after storage are illustrated in FIG. 23A-B and in TABLE 20. Both formulations showed similar supercoiled peak areas after storage. 2M1SN showed a larger supercoiled peak area compared to 2MSN.

Storage stability: VM202 freeze-dried preparations for further stability studies with accelerated degradation were stored for a 10-week period at 5°C, 25°C and 40°C (TABLE 4). At each time point, samples were taken from storage at a given temperature and evaluated visually. Photographs of the vials were taken and samples were evaluated for pellet shape. Samples were dissolved in 5 ml filtered water and evaluated for clarity and precipitation. All lyophilized pellets remained intact and did not show any sign of change on storage regardless of temperature conditions. Cloudiness was observed in the dissolved 2MSN after storage for 10 weeks. 2M1SN was clear after storage. The appearance of each formulation after storage for 10 weeks at 5°C, 25°C and 40°C is shown in FIG. 24A-C.

Analyzes of concentration, turbidity, moisture content and analysis by capillary electrophoresis were performed to assess the stability of lyophilized VM202 during temperature stresses.

At each time point, the samples were dissolved and analyzed for absorbance at 260 nm to determine the concentration of VM202 in the vials. TABLE 21 shows that the concentration of VM202 in lyophilized samples after 10 weeks of storage at a given temperature is similar to the results obtained at T equal to 0. Therefore, temperature stress did not induce large changes in the concentration of VM202.

The turbidity of each sample was also evaluated at each time point for two (2) different wavelengths. While the operators detected visible haze in the samples, no significant difference in haze was observed when measured at 650 nm. Therefore, it was determined that 450 nm was adequate to assess differences in turbidity between preparations. The results from this evaluation are detailed in TABLE 22. 2MSN exhibited moderate absorbance at 450 nm, regardless of storage temperature conditions. 2M1SN showed low absorption at 450 nm at all temperatures.

Moisture content analysis was also performed at each time point. TABLE 23 describes the results regarding moisture content after 10 weeks of storage at a given temperature. After 10 weeks of storage at 5°C, 25°C and 40°C, similar moisture content was observed in all samples, except for 2M1SN stored at 5°C, which showed a slightly increased moisture content (1.56%).

Capillary electrophoresis analysis was used to monitor product purity. CE chromatograms and peak area values after 10 weeks of incubation at 40° are shown in FIG. 25 and in TABLE 24, respectively. Both preparations showed significant decreases in supercoiled peak area with corresponding increases in open ring peak compared to T=0 after dissolution after 10 weeks of storage at 40°C. 2M1SN showed the largest supercoiled peak area, although this value was significantly lower at 10 weeks of storage at 40°C than at zero. The highest open ring peak area levels were observed in 2 MS N.

Trends in CE-determined supercoiled peak purity over 10 weeks at different temperatures are detailed in FIG. 26A-C - at 5°C (Fig. 26A), at 25°C (Fig. 26B) or at 40°C (Fig. 26C). Significant differences in purity were observed after incubation at elevated temperature. As illustrated in TABLE 24, 2M1SN containing 1% sucrose and 0.45% NaCl showed the highest level of purity after storage at 40°C.

Trends in open ring impurity peaks detected by CE during storage at a given temperature are shown in FIG. 27A-C - at 5°C (Fig. 27A), at 25°C (Fig. 27B) or at 40°C (Fig. 27C). These graphs show increases in the open ring peak corresponding to storage at an elevated storage temperature. After incubation at 40° C., a higher level of open ring peak contaminants was observed for 2MSN. 2M1SN showed a smaller open ring peak at all temperatures, but followed the same trend as 2MSN.

6.8.1.5. Summary of analysis results

Prior to freeze-drying, all analyzed preparations containing plasmid DNA (VM202) remained clear, with no discoloration or visible particles. In addition, the results of capillary electrophoresis (CE) consistently demonstrated the high purity of all these compositions.

The first and second rounds of small scale lyophilization assays were performed for VM202 using six (6) different formulations (KP8M2SN, KP8MS3N(equal to 2MSN), KP8MT3N, 4MSN, 3MSN and control) where the product was filled in a volume of 0.75 ml in 3 ml glass vials. An accelerated degradation stability study was performed on VM202 using the same four (4) preparations (4MSN, 3MSN, 2MSN and control) of the second round of lyophilization on a small scale, filled in a volume of 5 ml into 20 ml glass vials, and lyophilization parameters were determined from the data obtained from small scale lyophilization studies. Also, two (2) other formulations (2MSN and 2M1SN) were analyzed in an additional accelerated degradation stability study based on data from the original accelerated degradation stability study. These cycles of lyophilization were developed from the results obtained after DSC analysis under conditions close to ambient conditions. An annealing step was included in the lyophilization cycle because it was determined that the devitrification temperature was eliminated after annealing.

After lyophilization, first-class or good precipitation was observed for all compositions, with the exception of the control containing 0.9% NaCl and 1.1% sucrose. The control is the formulation previously described in US Pat. No. 8,389,492, which is incorporated herein by reference in its entirety.

However, upon dissolution of the lyophilized compositions, all compositions analyzed except for 2M1SN (10 mM potassium phosphate, pH 8.0, 2% mannitol, 1% sucrose, 0.45% NaCl) were cloudy. This turbidity remained even after storage of the dissolved compositions at 25°C for 3 and 7 days after dissolution. Only 2M1SN remained clear after dissolution and subsequent storage at 25°C for 3 and 7 days after dissolution, and there was no change in VM202 concentrations in 2M1SN throughout this study.

Capillary electrophoresis was effective in assessing the purity of the VM202 product. After lyophilization and dissolution, all preparations showed a decrease in the supercoiled peak area. A decrease in supercoiled peak area was also observed in all preparations after 10 weeks of storage at 25°C and 40°C. However, 2M1SN exhibited the highest percentage of supercoiled peak corresponding to the smallest percentage increase in open ring peak over the 10 week stability study.

These results indicate that lyophilized VM202 retains first-class pellet appearance and the highest level of purity (supercoiled DNA) after lyophilization and storage when prepared at 0.5 mg/mL with 10 mM potassium phosphate, pH 8.0, 2% mannitol, 1% sucrose, 0.45% NaCl in a 5 ml fill volume in 20 ml vials (2M1SN in TABLE 25). Although the index of instability increases for VM202 at higher temperatures, less degradation was observed in this preparation of 2M1SN than in other analyzed preparations.

6.8.2. Example 2 VM202 Lyophilized Composition (Study 002)

Various VM202-containing lyophilized preparations were prepared and analyzed to test the quality of VM202 in the drug product formulation after lyophilization, as well as the quality of VM202 in preparations with slight variations in pH and/or concentrations of excipients and stabilizers.

6.8.2.1. Scheme of experiments

Materials: The active pharmaceutical ingredient (API) tested in this study was VM202. The substance used for this study contained the following: The reagents and substances used to prepare and analyze VM202 were as follows:

Preparation parameter: in this study, the following parameters were recorded:

(1) Fill volume: 5ml

(2) API concentration: 0.5mg/ml

(3) Buffer concentration: 10mM potassium phosphate

(4) Sucrose concentration: 1%

In these preparations, the following preparation parameters were checked:

(1) pH: 7.0; 8.0 and 9.0

(2) Mannitol concentration: 1% and 2%

(3) Sodium chloride concentration: 0.45%, 0.6% and 0.9%

The preparation listed below in TABLE 27 was prepared for analysis:

6.8.2.2. Receiving drugs

6.8.2.2.1. Research on a small scale

A small scale analysis was performed to determine the pH values of the buffer required to dilute the drug substance (DS) to reach the drug target pH values in TABLE 27. A small scale dilution study was performed with placebo to preserve the drug substance. as 0.9% NaCl (same formulation as for DS) instead of DS. A 0.9% NaCl placebo was prepared without pH adjustment to better represent the composition of the DS formulation. The dilution buffer composition (TABLE 28) was calculated based on a dilution of DS from 1.6 mg/mL to 0.5 mg/mL. Small-scale analysis revealed that after dilution with formulation buffers, all final formulations exhibited pH values at or near the target values in TABLE 27. However, since the pH of formulation F3 (9.0) was outside the phosphate buffering range, pH slip was observed. to smaller values. To best compensate for this, a dilution buffer with a higher pH (9.5) was chosen. Small scale testing showed a pH of 9.2 immediately after dilution followed by a decrease in pH to 9.1 and 8.5 after 24 and 48 hours, respectively.

To best achieve the pH targets for this study, formulations were prepared within one day of lyophilization and pH changes monitored after dissolution. The dilution buffers shown in TABLE 28 were used to prepare the preparation.

6.8.2.2.2. Lyophilization

VM202 at 1.6 mg/mL in 0.9% NaCl supplied from Cobra was removed from storage at -70°C and thawed at 5°C overnight. 35 ml of DS was diluted in 21 ml of dilution buffers (TABLE 28) to a concentration of 1.0 mg/ml. After dilution, pH and API concentration were measured. Despite small-scale placebo assay results, these formulations did not produce pH targets in the presence of VM202, so the pH of the stock dilution buffers was further adjusted during dilution of 1.0 mg/ml DS in 48 ml dilution buffer (TABLE 29) to a final concentration of 0.5 mg/ml.

After dilution, the preparations were subjected to sterilizing filtration using a 0.2 μm filter based on cellulose acetate, filled into 20 ml glass, aspirated vials with a 5 ml filling volume (2.5 mg dose) in a BSC (microbiological safety box). After filling, the vials were partially stoppered and loaded into a lyophilizer for freeze drying. Empty vials were used to completely surround vials containing DS VM202. After lyophilization, these vials were completely sealed inside the lyophilization chamber with a partial vacuum of 600 Torr before removal. These vials were closed with crimp caps, labeled and placed at -70°C. One (1) vial of each formulation was placed at 5°C. One (1) vial per formulation was diluted with 5 ml Milli-Q filtered water and analyzed using samples prior to lyophilization.

6.8.2.2.3. Methods of analysis

Lyophilized preparations were analyzed at different time points, as summarized in TABLE 30.

Visual inspection: A visual inspection was performed under a white light source (13 W fluorescent tube) against a black and white background. We received digital photographs of all preparations.

Concentration measurement (A260): Spectrophotometric analysis was performed with a 1 cm pathlength quartz cuvette by means of a Beckman Coulter DU800. The concentration of plasmid DNA of the test samples was determined by measuring the absorbance at 230, 260, 280 and 350 nm. Concentration analyzes were performed using UV/Vis protocol. by Helixmith. The calculations were carried out using the following equations:

Concentration (µg/ml)=[(D-E)/C] × (V/A)

Yield=(low µg/mL) / (high µg/mL) × 100%

OD _260nm / OD _280nm =D / F

OD _260nm / OD _230nm =D / G

Where:

A is the mass of the sample taken for breeding

B is the total weight of the sample and buffer used for dilution

C - extinction coefficient of a 0.005% solution in a cuvette with a path length of 1 cm (0.02)

D is the optical density for the maximum at 260 nm. E - absorbance measured at 350 nm

F is the absorbance measured at 280 nm

G is the absorbance measured at 230 nm

Acceptance Criteria:

D must fall within the range of 0.5 to 1.5 OD units

Yield must be 98% or more

OD _260nm / OD _280nm - should be between 1.8 and 2.0

OD _260nm / OD _230nm - must be 1.1 or more

pH: pH analysis was performed using a SympHony® pH meter (VWR Scientific, catalog # SB70P) calibrated with three pH standards (pH 4, 7, and 10) with a calibration slope of 95% or greater. The samples were not temperature corrected, equilibrated at ambient temperature, and measured.

Haze measurement (A350, A450, A650): Haze was determined by measuring sample absorbance at 350, 450, and 650 nm using a Beckman Coulter DU800. Generally, preparations with A650 greater than 0.01 are considered to exhibit increased turbidity.

Capillary zonal electrophoresis: Before analysis, the capillary was conditioned by passing Milli-Q water, 0.1 N. NaOH, 0.1 N. HCl and again Milli-Q water, each at 137.9 kPa for 10 minutes (conditioning only took 40 minutes). 40 μl of the sample at a concentration of 0.5 mg/ml was loaded into a polyethylene insert for analysis.

Instrument: Beckman Coulter PA 800+ CE (S/N 3063309)

Capillary: neutral coated capillary, inner diameter 50 µm, total length 40 cm, effective length 30 cm, aperture 8 µm (Beckman Coulter P/N 477441, lot no. M812134)

Data Analysis: 32 Karat (version 9.0)

Injection pressure/time: 3.447 kPa; 60 s

Separation buffer/pressure: 100 mM phosphoric acid (pH 2.60), 85%/137.9 kPa; 60 s

Wash buffer/pressure: Milli-Q water / 137.9 kPa; 60 s

Detection: UV at 254 nm

Separation voltage: 17 kV; linear change - 0.17 min; 14 min.

Residual Moisture Content: Karl Fisher Coulometer C20 (Mettler Toledo) was used for moisture analysis. An Apura 1% water standard oven was used to determine the accuracy of the system. The sample vials were brought to room temperature before removing the assay caps. Approximately 100 mg of substance was used for each assay.

6.8.2.3. Summary of analysis results

This section summarizes the quality of lyophilized VM202 in different formulations.

6.8.2.3.1. Lyophilization Cycle Profile

A previously developed lyophilization cycle was used to lyophilize 2.5 mg doses of VM202 in various formulations. Ramping the freeze temperature from -50°C to -20°C and holding for two (2) hours before applying vacuum allowed annealing to occur. The annealing process helps the crystallization of amorphous excipients. Due to the unique characteristic of the lyophilizer used in these experiments, which can terminate the program if the vacuum does not reach the set point within these time frames, a step was used at the start of primary drying where a vacuum of 100 mTorr (13.33 Pa) is initially applied, and it is adjusted to the final vacuum as the primary drying begins. Primary drying is carried out at a shelf temperature of -20°C (about 53.4 hours) with a chamber pressure of 50 mTorr (6.67 Pa) (TABLE 31). Secondary drying at a shelf temperature of 20°C is designed to remove residual water that did not sublimate during the primary drying step.

* Primary drying time changed from 3240 min to 3200 min due to program limitations. The readings of the Pirani manometer and the capacitance manometer were merged before proceeding to the secondary drying.

On FIG. 28 shows a graph of the entire lyophilization cycle. The temperature of the product dropped to about -40° C. due to heat loss from sublimation when the vacuum was applied. The value of the Pirani manometer merged with the reading of the capacitance manometer at approximately 41 hours, which confirmed the completion of the primary drying process. After 62 hours of the whole cycle, secondary drying was initiated at 20° C. and the samples were allowed to dry for another 13 hours. The entire cycle lasted approximately 75 hours.

The measured moisture content of precipitation is shown in TABLE 32.

6.8.2.3.2. Lyophilization Study

Visual check: After lyophilization, F1, F2 and F3 showed good precipitation with only slight drying. F5 showed significant drying of the sediment. Complete collapse of the precipitates was observed for F4 (FIG. 32) and F6 (FIG. 34). Dissolved liquid samples F1 (Fig. 29), F2 (Fig. 30), F3 (Fig. 31), F4 (Fig. 32) and F5 (Fig. 33) were clear, colorless and free of visible particles, comparable to control before lyophilization. After dissolution, F6 was cloudy.

Concentration (A ₂₆₀ ): all samples had their target concentrations before lyophilization and after dissolution, as shown in TABLE 33.

pH and Dissolution Time: Due to the buffering limitation of the F3 preparation, the pH of F3 was not stable and decreased rapidly during filling. By the time samples were analyzed prior to lyophilization, it had already decreased from pH 9.0 to 8.7. A further decrease in pH value was observed after dissolution (Δ equal to -0.4) compared to the control before lyophilization. After dissolution, preparations F1, F2, F4, F5, F6 showed the same pH values (Δ less than or equal to 0.1) compared to their pre-lyophilization controls. Analysis of the dissolution time showed that the lyophilized precipitate was dissolved after 1-1.5 minutes. Results for pH and dissolution time are detailed in TABLE 34.

Haze (A ₃₅₀ , A ₄₅₀ , A ₆₅₀ ): small increases in haze were observed for F6 at 350 nm, 450 nm and 650 nm after dissolution. All other preparations did not show significant turbidity before lyophilization or after dissolution, as shown below in TABLE 35.

Note: Water absorption was subtracted to obtain haze values for samples with an A ₆₅₀ greater than or equal to 0.01 considered cloudy.

Capillary Zone Electrophoresis (CZE): Dissolved samples were further checked for product purity by CZE. F6 was not analyzed due to turbidity. Electrophoregrams and results in tabular form from CZE are detailed in FIG. 35 and in TABLE 36, respectively. After dissolution, all formulations showed slight increases in the open ring peak with corresponding decreases in the supercoiled peak (97.5%-99.0%) compared to pre-lyophilization samples (99.1%-100.0%). F4 and F5 showed the smallest percentage increase in open ring peak less than or equal to 0.4% compared to the other formulations. With the exception of preparations with poor sediment appearance, the present preparation (F1) showed the highest purity before (100%) and after lyophilization (98.6%).

Conclusions: The purpose of this study was to assess the quality of VM202 in a drug product formulation - 0.5 mg/ml VM202 in 10 mM potassium phosphate with 2% mannitol, 1% sucrose, 0.45% NaCl at pH 8.0, followed by lyophilization, and the quality of VM202 in formulations with slight variations in pH and/or concentrations of excipients and stabilizers from the drug product formulation. The analytical methods used in this study included capillary zone electrophoresis (CZE), visual inspection for sediment clarity and appearance, turbidity, concentration (A260), pH, and moisture content (Karl Fischer) analyzes, which all have been shown to be effective stability-indicating product assays.

In this study, lyophilized VM202 was evaluated at a dose of 2.5 mg per vial in a 5 ml fill volume. Small scale pH analysis was performed with 0.9% NaCl placebo to establish the correct preparation procedures for the larger scale VM202 dilution method.

Prior to lyophilization, all VM202 preparations were clear with no discoloration, contained no visible particles, and were at the target concentration. The pH of the 10 mM potassium phosphate preparation of 9.0 was not stable and decreased rapidly during filling, with further decreases observed after lyophilization and dissolution. The capillary zone electrophoresis results for all preparations before lyophilization showed high purity, with the drug product preparation showing the highest percentage of supercoiled peak.

Good precipitation was observed for the drug product preparation and drug product preparations at higher and lower pH after lyophilization. After dissolution, the preparation with the highest concentration of NaCl showed turbidity, while the remaining preparations were clear, colorless and did not contain visible particles, comparable to the control before lyophilization. After dissolution, all formulations showed slight increases in the open ring peak with corresponding decreases in the supercoiled peak. Excluding formulations with poor precipitate appearance, the lyophilized drug product formulation exhibited the highest purity as determined by capillary zonal electrophoresis.

The results obtained from this study indicate that lyophilized VM202 retains good pellet appearance and the highest level of purity (supercoiled DNA) after lyophilization at 0.5 mg/mL with 10 mM potassium phosphate at pH 8.0, 2% mannitol, 1% sucrose, 0.45% NaCl in a 5 ml fill volume in 20 ml vials.

6.8.3. Example 3 pTx-IGF-1X10 Lyophilized Composition

Various lyophilized formulations containing pTx-IGF-1X10 were prepared and analyzed for the quality of pTx-IGF-1X10 in the post-lyophilization formulation. For analysis received drugs listed below in TABLE 37.

6.8.3.1. Summary of analysis results

Visual inspection: After lyophilization, F1, F2, F7, F8 and F9 showed good precipitation with only slight drying. F5 showed significant drying of the sediment. F3 had a spattered look. For F4 and F6, complete destruction of sediments was observed. Dissolved liquid samples F1 (Fig. 36), F2 (Fig. 37), F3 (Fig. 38), F4 (Fig. 39), F5 (Fig. 40), F6 (Fig. 41), F7 (Fig. 42) ), F8 (FIG. 43) and F9 (FIG. 44) were clear, colorless and free of visible particles - comparable to pre-lyophilization control. To determine the turbidity (clarity) of the samples, the samples were compared to a 4000NTU I, II, III, IV turbidity calibration standard. A preparation with "clarity less than 1" may be judged to be unclouded.

Concentration (A260): All samples had their target concentrations before lyophilization and after dissolution as shown in TABLE 38 below.

pH and dissolution time: The pH value of F3 decreased from pH 9.0 to 7.6 after dissolution. After dissolution, preparations F1, F2, F4, F5, F6, F7, F8, F9 showed the same pH values (Δ less than or equal to 0.5) compared to their pre-lyophilization controls. Dissolution time analysis showed that the lyophilized pellet dissolved from 1 min 20 s to 4 min 05 s. Results for pH and dissolution time are detailed in TABLE 39.

Capillary Electrophoresis (CE): Dissolved samples were further checked for product purity by CE. Electrophoregrams and results in tabular form from CE are detailed in FIG. 45 and in TABLE 40, respectively. After dissolution, all preparations, except for F3, showed small increases (0.5-3%) in the open ring peak with corresponding decreases in the superspring peak compared to the preparations before lyophilization. With the exception of formulations with poor pellet appearance, the present formulation (F1) showed the smallest reduction in supercoiled peak after lyophilization.

6.8.4. Example 4 Lyophilized composition pCK-SDF-1α

6.8.4.1. Experiment scheme

Various lyophilized preparations containing pCK-SDF-1α were obtained and analyzed for the quality of pCK-SDF-1α in the preparation after lyophilization. For analysis received drugs listed below in TABLE 41.

6.8.4.2. Summary of analysis results

Visual inspection: after lyophilization, F1, F2, F3, F7, F8 and F9 showed good precipitation with only slight drying. F5 showed significant drying of the sediment. Complete destruction of sediments was observed for F4 and F6. Dissolving liquid samples F1 (Fig. 46), F2 (Fig. 47), F3 (Fig. 48), F4 (Fig. 49), F5 (Fig. 50), F6 (Fig. 51), F7 ( Fig. 52), F8 (Fig. 53) and F9 (Fig. 54) were clear, colorless and free of visible particles, comparable to pre-lyophilization control. To determine the turbidity (clarity) of the samples, the samples were compared to a 4000NTU I, II, III, IV turbidity calibration standard. A preparation with "clarity less than I" may be judged to be unclouded.

Concentration (A260): All samples were at their target concentrations before lyophilization and after reconstitution as shown in TABLE 42 below.

pH and dissolution time: The pH value of F3 decreased from pH 9.0 to 7.9 after dissolution. After dissolution, preparations F1, F2, F4, F5, F6, F7, F8, F9 showed the same pH values (Δ less than or equal to 0.5) compared to their pre-lyophilization controls. Dissolution time analysis showed that the lyophilized pellet dissolved from 1.5 minutes to 4 minutes. Results for pH and dissolution time are detailed below in TABLE 43.

Capillary electrophoresis: dissolved samples were further checked for product purity by CE. Electrophoregrams and results in tabular form from CE are detailed in FIG. 55 and in TABLE 44, respectively. After dissolution, all preparations showed slight increases (0.5-2%) in the open ring peak with corresponding decreases in the supercoiled peak compared to the pre-lyophilization preparations. With the exclusion of preparations with poor sediment appearance, the present preparation (F1) showed the smallest decrease in the supercoiled peak after lyophilization.

7. Inclusion by link

All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually identified as incorporated by reference for all purposes.

8. Equivalents

While various specific embodiments have been illustrated and described, the above description of the invention is not intended to be limiting. It will be understood that various changes can be made without departing from the spirit and scope of the invention(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Sequence Listing:

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SEQUENCE LIST

<110> HELIXMITH CO., LTD.

<120> LYOPHILIZED PHARMACEUTICAL COMPOSITIONS FOR NAKED DNA GENOTHERAPY

<130> 33730-40319/WO (014WO)

<140>

<141>

<150> 62/700.655

<151> 2018-07-19

<160> 36

<170> PatentIn version 3.5

<210> 1

<211> 728

<212> Protein

<213> Homo sapiens

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Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu

1 5 10 15

Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln

20 25 30

Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr

35 40 45

Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val

50 55 60

Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu

65 70 75 80

Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys

85 90 95

Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe

100 105 110

Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys

115 120 125

Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys

130 135 140

Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His

145 150 155 160

Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr

165 170 175

Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser

180 185 190

Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu

195 200 205

Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp

210 215 220

His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro

225 230 235 240

His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp

245 250 255

Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr

260 265 270

Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys

275 280 285

Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu

290 295 300

Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile

305 310 315 320

Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu

325 330 335

His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn

340 345 350

Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr

355 360 365

Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp

370 375 380

Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met

385 390 395 400

Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp

405 410 415

Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala

420 425 430

Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His

435 440 445

Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys

450 455 460

Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu

465 470 475 480

Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val

485 490 495

Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg

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Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp

515 520 525

Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr

530 535 540

Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys

545 550 555 560

Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly

565 570 575

Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp

580 585 590

Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu

595 600 605

Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn

610 615 620

Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu

625 630 635 640

Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu

645 650 655

Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp

660 665 670

Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu

675 680 685

Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly

690 695 700

Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile

705 710 715 720

Leu Thr Tyr Lys Val Pro Gln Ser

725

<210> 2

<211> 723

<212> Protein

<213> Homo sapiens

<400> 2

Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu

1 5 10 15

Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln

20 25 30

Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr

35 40 45

Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val

50 55 60

Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu

65 70 75 80

Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys

85 90 95

Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe

100 105 110

Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys

115 120 125

Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys

130 135 140

Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His

145 150 155 160

Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg

165 170 175

Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg

180 185 190

Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr

195 200 205

Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly

210 215 220

Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys Phe

225 230 235 240

Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg

245 250 255

Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro His

260 265 270

Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr Met

275 280 285

Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln

290 295 300

Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro

305 310 315 320

Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro

325 330 335

Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn Pro

340 345 350

Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile Arg

355 360 365

Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly Gln

370 375 380

Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser Gln

385 390 395 400

Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp

405 410 415

Leu His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr

435 440 445

Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys

450 455 460

Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val Ile

465 470 475 480

Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro Thr

485 490 495

Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys His

500 505 510

Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala Arg

515 520 525

Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly

530 535 540

Ile His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu

545 550 555 560

Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu

565 570 575

Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr Ile

580 585 590

Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys Ser

595 600 605

Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu Leu

610 615 620

Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln His

625 630 635 640

His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala

645 650 655

Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu

660 665 670

Val Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro

675 680 685

Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val

690 695 700

Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys Val

705 710 715 720

Pro Gln Ser

<210> 3

<211> 482

<212> DNA

<213> Homo sapiens

<400> 3

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

ag 482

<210> 4

<211> 1675

<212> DNA

<213> Homo sapiens

<400> 4

cctacaggaa aactactgtc gaaatcctcg aggggaagaa gggggaccct ggtgtttcac 60

aagcaatcca gaggtacgct acgaagtctg tgacattcct cagtgttcag aagttgaatg 120

catgacctgc aatggggaga gttatcgagg tctcatggat catacagaat caggcaagat 180

ttgtcagcgc tgggatcatc agacaccaca ccggcacaaa ttcttgcctg aaagatatcc 240

cgacaagggc tttgatgata attattgccg caatcccgat ggccagccga ggccatggtg 300

ctatactctt gaccctcaca cccgctggga gtactgtgca attaaaacat gcgctgacaa 360

tactatgaat gacactgatg ttcctttgga aacaactgaa tgcatccaag gtcaaggaga 420

aggctacagg ggcactgtca ataccatttg gaatggaatt ccatgtcagc gttgggattc 480

tcagtatcct cacgagcatg acatgactcc tgaaaatttc aagtgcaagg acctacgaga 540

aaattactgc cgaaatccag atgggtctga atcaccctgg tgttttacca ctgatccaaa 600

catccgagtt ggctactgct cccaaattcc aaactgtgat atgtcacatg gacaagattg 660

ttatcgtggg aatggcaaaa attatatggg caacttatcc caaacaagat ctggactaac 720

atgttcaatg tgggacaaga acatggaaga cttacatcgt catatcttct gggaaccaga 780

tgcaagtaag ctgaatgaga attactgccg aaatccagat gatgatgctc atggaccctg 840

gtgctacacg ggaaatccac tcattccttg ggattattgc cctatttctc gttgtgaagg 900

tgataccaca cctacaatag tcaatttaga ccatcccgta atatcttgtg ccaaaacgaa 960

1020 acaattgcga gttgtaaatg ggattccaac

gagatacaga aataaacata tctgcggagg atcattgata agggagtt gggttcttac 1080

tgcacgacag tgtttccctt ctcgagactt gaaagattat gaagcttggc ttggaattca 1140

tgatgtccac ggaagaggag atgagaaatg caaacaggtt ctcaatgttt cccagctggt 1200

atatggccct gaaggatcag atctggtttt aatgaagctt gccaggcctg ctgtcctgga 1260

tgattttgtt agtacgattg atttacctaa ttatggatgc acaattcctg aaaagaccag 1320

ttgcagtgtt tatggctggg gctacactgg attgatcaac tatgatggcc tattacgagt 1380

ggcacatctc tatataatgg gaaatgagaa atgcagccag catcatcgag ggaaggtgac 1440

tctgaatgag tctgaaatat gtgctggggc tgaaaagatt ggatcaggac catgtgaggg 1500

ggattatggt ggcccacttg tttgtgagca acataaaatg agaatggttc ttggtgtcat 1560

tgttcctggt cgtggatgtg ccattccaaa tcgtcctggt atttttgtcc gagtagcata 1620

ttatgcaaaa tggatacaca aaattatttt aacatataag gtaccacagt catag 1675

<210> 5

<211> 3756

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 5

cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60

atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120

cgccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180

tagggacttt ccattgacgt caatggggtgg agtatttacg gtaaactgcc cacttggcag 240

tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc 300

360

acgtattagt catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg 420

gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480

tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga 540

cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga 600

accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg 660

accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga 720

gtgacgtaag taccgcctat agactctata ggcacacccc tttggctctt atgcatgcta 780

tactgttttt ggcttggggc ctatacaccc ccgcttcctt atgctatagg tgatggtata 840

gcttagccta taggtgtggg ttattgacca ttattgacca ctcccctatt ggtgacgata 900

ctttccatta ctaatccata acatggctct ttgccacaac tatctctatt ggctatatgc 960

caatactctg tccttcagag actgacacgg actctgtatt tttacaggat ggggtcccat 1020

ttattattta caaattcaca tatacaacaa cgccgtcccc cgtgcccgca gtttttatta 1080

aacatagcgt gggatctcca cgcgaatctc gggtacgtgt tccggacatg ggctcttctc 1140

cggtagcggc ggagcttcca catccgagcc ctggtcccat gcctccagcg gctcatggtc 1200

gctcggcagc tccttgctcc taacagtgga ggccagactt aggcacagca caatgcccac 1260

caccaccagt gtgccgcaca aggccgtggc ggtagggtat gtgtctgaaa atgagctcgg 1320

agattgggct cgcaccgctg acgcagatgg aagacttaag gcagcggcag aagaagatgc 1380

aggcagctga gttgttgtat tctgataaga gtcagaggta actcccgttg cggtgctgtt 1440

aacggtggag ggcagtgtag tctgagcagt actcgttgct gccgcgcgcg ccaccagaca 1500

taatagctga cagactaaca gactgttcct ttccatgggt cttttctgca gtcaccgtcc 1560

ttgacacgaa gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattctgcag 1620

atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc 1680

ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 1740

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 1800

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 1860

ggattgggaa gacaatagca ggcatgctgg ggagtcgaaa ttcagaagaa ctcgtcaaga 1920

aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag 1980

cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc 2040

tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt 2100

tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg 2160

ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg 2220

tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga 2280

2340

gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc 2400

cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca 2460

gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt 2520

tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac 2580

agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat 2640

agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga 2700

aacgatcctc atcctgtctc ttgatcagat cttgatcccc tgcgccatca gatccttggc 2760

ggcaagaaag ccatccagtt tactttgcag ggcttcccaa ccttaccaga gggcgcccca 2820

gctggcaatt ccggttcgct tgctgtccat aaaaccgccc agtctagcta tcgccatgta 2880

agccctgc aagctacctg ctttctcttt gcgcttgcgt tttcccttgt ccagatagcc 2940

cagtagctga cattcatccg gggtcagcac cgtttctgcg gactggcttt ctacgtgaaa 3000

aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 3060

tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 3120

tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 3180

ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 3240

ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta 3300

gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 3360

3420

ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 3480

agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 3540

aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 3600

aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 3660

3720

cggttcctgg ccttttgctg gccttttgct cacatg 3756

<210> 6

<211> 4956

<212> DNA

<213> Homo sapiens

<400> 6

gtaagaacag tatgaagaaa agagatgaag cctctgtctt ttttacatgt taacagtctc 60

atattagtcc ttcagaataa ttctacaatc ctaaaataac ttagccaact tgctgaattg 120

tattacggca aggtttatat gaattcatga ctgatattta gcaaatgatt aattaatatg 180

ttaataaaat gtagccaaaa caatatctta ccttaatgcc tcaatttgta gatctcggta 240

tttgtgaaat aataacgtaa acttcgttta aaaggattct tcttcctgtc tttgagaaag 300

tacggcactg tgcaggggga gaggttgatt gtgaaaaatc agaggtagat gagaatctta 360

ctgaggggctg agggttcttt aaccttggtg gatctcaaca ttggttgcac attaaaatca 420

cctgctgcaa gcccttgacg aatcttactt agaagatgac aacacagaac aattaaatca 480

gaatctctgg ggagaatagg gcaccagtat tttttgagct cccaccatga ttccaaagtg 540

cagccaaatt tgagaaccac tgctaaaagc tcaagcttca gattgaccag cttttccatc 600

tcacctatcg cctaaagacc aaattggata aatgtgttca ttacgacaga tgggtactat 660

ttaaagatga gtaaacacaa tatacttagg ctcgtcagac tgagagtttt aatcatcact 720

gaggaaaaac atagatatct aatactgact ggagtattag tcaaggctta tttcacacac 780

aattttatca gaaaccaaag tagtttaaaa cagctctccc cttattagta atgcattgga 840

gggtttactt taccatgtac cttgctgagc actgtacctt gttaatctca tttacttgta 900

atgagaacca cacagcgggt agttttattg gttctatttt acctacatga caaaactgaa 960

gcataaaaac acttagtaag ttttcagtgt catgcacaac taggaagtga catggccaga 1020

atataagccc agtcaccatc actctataac ctgcgctttt aacaacttca gggcatgaca 1080

catttggccg gtcagtagaa cccatgctgt gatttgtttt tgcagtggtg gtgatgactg 1140

ccttgttgaa tccacttttt attctattcc attttgggga cacaattctg caagatgatt 1200

1260

ccacactggg attaaaccta tgatcttggc ctaattaaca ctagctagta agtgtccaag 1320

1380

caattatgta gaaatgcctc taaaacacag tattttcctt atattacaaa aacaaaaatt 1440

ataattggtt ttgtcctctt ttgagagttt gcatggtgtt actccctgca tagtgaagaa 1500

aacattttat ttaagtagat ggatctaagt ttttcatgaa caaaggaatg acatttgaaa 1560

tcaatcctac cctagtccag gagaatgcat tagattaacc tagtagaggt cttatttcac 1620

1680

gggaactggc ttcctagtcc aatcagctct tttaccaatg aacacttcct tgtgatatag 1740

atgttttgg ccgagaggat ccagtatatt aataaaatcc ctttttgtat tcaatgaggg 1800

aaacacataa ttttcatcaa ttagcagctt attggaatat ctgcatgatg gtttaacact 1860

tttaagtgtt gactaaagat taattttaca gaaaatagaa aaagaaatat gtttctgtct 1920

ggaggaatga tttattgttg acccctaaat tgaaatattt tactagtggc ttaatggaaa 1980

gatgatgaaa gatgatgaaa ttaatgtaga agcttaacta gaaaatcagg tgacctgata 2040

tctacatctg tatccttcat tggccaccca gcattcatta atgaatcaga tgatggaata 2100

2160

gaagacctag tttatatttc aaagtatatt tggatgtaac ccaattttaa acatttcctc 2220

acttgtctct cttaaagcct tgccaacagc aaggacagag aaccaaaaat agtgtatata 2280

tgaataaatg cttattacag aatctgctga ctggcacatg ctttgtgtgt aatgggttct 2340

cataaacact tgttgaatga acacacataa gtgaaagagc atggctaggc ttcatccctt 2400

ggtcaaatat ggggtgctaa agaaaagcag gggaaataca ttgggacact aacaaaaaaa 2460

aacagttaat ttaggtaaaa gataaaatac accacagaat gaagaaaaga gatgacccag 2520

actgctcttt aaccttcatg tcctagagag gtttttgata tgaattgcat tcagaattgt 2580

ggaaaggagc ccatcttttc tcttcatttt gattttatta actccaatgg gggaatttta 2640

ttcgtgtttt ggccatatct acttttgatt tctacattat tctctcttcc tttctacctg 2700

tatttgtcct aataaattgt tgacttatta attcactact tcctcacagc ttttttttgg 2760

2820

tgtagagggg acaaaaatcc tctctcaaac tataaatatt gagtatttgt gtattgaaca 2880

tttgctataa ctactaggtt tcttaaataa tcttaatata taaaatgata tagaaaaagg 2940

gaaattatag ttcgtattat tcatctaagt gaagagatta aaacccaggg agtaaataaa 3000

ttgtctaagg actaaggttg tatactattt aggtgataga tatggggcaa ccgtatggggt 3060

tttatgatta acaaataaac ttctcaccac tctaccatat caacttttcc ataaaagaga 3120

gctatagtat tctttgctta aataaatttg attagtgcat gacttcttga aaacatataa 3180

agcaaaagtc acatttgatt ctatcagaaa agtgagtaag ccatggccca aacaaaagat 3240

3300

ttactgttag gaattgtgaa attatgctga attttagttg cattataatt tttgtcagtc 3360

atacggtctg acaacctgtc ttatttctat ttccccatat gaggaatgct agttaagtat 3420

ggatattaac tattactact tagatgcatt gaagttgcat aatatggata atacttcact 3480

ggttccctga aaatgtttag ttagtaataa gtctcttaca ctatttgttt tgtccaataa 3540

tttatatttt ctgaagactt aactctagaa tacactcatg tcaaaatgaa agaatttcat 3600

3660

3720

atactaaaat ccttggactt cactctaatt ttagtgccat ttagaactca aggtctcagt 3780

aaaagtagaa ataaagcctg ttaacaaaac acaaactgaa tattaaaaat gtaactggat 3840

3900

tgtaaagtct ggcagacaaa tgcaatatct aattgttgag tccaatatca caagcagtac 3960

aaaagtataa aaaagacttg gccttttcta atgtgttaaa atactttatg ctggtaataa 4020

cactaagagt agggcactag aaattttaag tgaagataat gtgttgcagt tactgcactc 4080

aatggcttac tattataaac caaaactggg atcactaagc tccagtcagt caaaatgatc 4140

aaaattattg aagagaataa gcaattctgt tctttattag gacacagtag atacagacta 4200

caaagtggag tgtgcttaat aagaggtagc atttgttaag tgtcaattac tctattatcc 4260

cttggagctt ctcaaaataa ccatataagg tgtaagatgt taaaggttat ggttacactc 4320

agtgcacagg taagctaata ggctgagaga agctaaatta cttactgggg tctcacagta 4380

agaaagtgag ctgaagtttc agcccagatt taactggatt ctgggctctt tattcatgtt 4440

acttcatgaa tctgtttctc aattgtgcag aaaaaagggg gctatttata agaaaagcaa 4500

4560

gtggcagcga tttctcagtt ctgtcctaag tggccttgct caatcacctg ctatctttta 4620

gtggagcttt gaaattatgt ttcagacaac ttcgattcag ttctagaatg tttgactcag 4680

caaattcaca ggctcatctt tctaacttga tggtgaatat ggaaattcag ctaaatggat 4740

gttaataaaa ttcaaacgtt ttaaggacag atggaaatga cagaatttta aggtaaaata 4800

4860

gtaaaattaa taggcgaaaa aaagttgcat gctcttatac tgtaatgatt atcattttaa 4920

aactagcttt ttgccttcga gctatcgggg taaaga 4956

<210> 7

<211> 7113

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 7

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgaa ataataacgt aaacttcgtt taaaaggatt cttcttcctg tctttgagaa 780

agtacggcac tgtgcagggg gagaggttga ttgtgaaaaa tcagaggtag atgagaatct 840

tactgagggc tgagggttct ttaaccttgg tggatctcaa cattggttgc acattaaaat 900

cacctgctgc aagcccttga cgaatcttac ttagaagatg acaacacaga acaattaaat 960

cagaatctct ggggagaata gggcaccagt attttttgag ctcccaccat gattccaaag 1020

tgcagccaaa tttgagaacc actgctaaaa gctcaagctt cagattgacc agcttttcca 1080

tctcacctat cgcctaaaga ccaaattgga taaatgtgtt cattacgaca gatgggtact 1140

atttaaagat gagtaaacac aatatactta ggctcgtcag actgagagtt ttaatcatca 1200

ctgaggaaaa acatagatat ctaatactga ctggagtatt agtcaaggct tatttcacac 1260

acaattttat cagaaaccaa agtagtttaa aacagctctc cccttattag taatgcattg 1320

gagggtttac tttaccatgt accttgctga gcactgtacc ttgttaatct catttacttg 1380

taatgagaac cacacagcgg gtagttttat tggttctatt ttacctacat gacaaaactg 1440

aagcataaaa acacttagta agttttcagt gtcatgcaca actaggaagt gacatggcca 1500

gaatataagc ccagtcacca tcactctata acctgcgctt ttaacaactt cagggcatga 1560

cacatttggc cggtcagtag aacccatgct gtgatttgtt tttgcagtgg tggtgatgac 1620

1680

ttcttcatta ggaaacagag atgagtttatt gaccaacaca gaaagaaaaa gagtttgttg 1740

ctccacactg ggattaaacc tatgatcttg gcctaattaa cactagctag taagtgtcca 1800

agctgatcat ctctacaaca tttcaataac agaaaacaac aattttcaaa attagttact 1860

tacaattatg tagaaatgcc tctaaaacac agtattttcc ttatattaca aaaacaaaaa 1920

tttaattgg ttttgtcctc ttttgagagt ttgcatggtg ttactccctg catagtgaag 1980

aaaacatttt atttaagtag atggatctaa gtttttcatg aacaaaggaa tgacatttga 2040

aatcaatcct accctagtcc aggagaatgc attagattaa cctagtagag gtcttatttc 2100

accctgagtt ttctatgatc gtgattctct gctggaggag taattgtgaa atagatctct 2160

ctgggaactg gcttcctagt ccaatcagct cttttaccaa tgaacacttc cttgtgatat 2220

agatgtttat ggccgagagg atccagtata ttaataaaat ccctttttgt attcaatgag 2280

ggaaacacat aattttcatc aattagcagc ttattggaat atctgcatga tggtttaaca 2340

cttttaagtg ttgactaaag attaatttta cagaaaatag aaaaagaaat atgtttctgt 2400

ctggaggaat gatttattgt tgacccctaa attgaaatat tttactagtg gcttaatgga 2460

aagatgatga aagatgatga aattaatgta gaagcttaac tagaaaatca ggtgacctga 2520

tatctacatc tgtatccttc attggccacc cagcattcat taatgaatca gatgatggaa 2580

tagatcaagt ttcctaggaa cacagtgaat attaaaagaa aacaaaggga gcctagcacc 2640

tagagacct agtttatatt tcaaagtata tttggatgta acccaatttt aaacatttcc 2700

tcacttgtct ctcttaaagc cttgccaaca gcaaggacag agaaccaaaa atagtgtata 2760

tatgaataaa tgcttattac agaatctgct gactggcaca tgctttgtgt gtaatgggtt 2820

ctcataaaca cttgttgaat gaacacacat aagtgaaaga gcatggctag gcttcatccc 2880

ttggtcaaat atggggtgct aaagaaaagc aggggaaata cattgggaca ctaacaaaaa 2940

aaaacagtta atttaggtaa aagataaaat acaccacaga atgaagaaaa gagatgaccc 3000

3060

gtggaaagga gcccatcttt tctcttcatt ttgattttat taactccaat gggggaattt 3120

tattcgtgtt ttggccatat ctacttttga tttctacatt attctctctt cctttctacc 3180

3240

3300

tgtgtagagg ggacaaaaat cctctctcaa actataaata ttgagtattt gtgtattgaa 3360

3420

gggaaattat agttcgtatt attcatctaa gtgaagagat taaaacccag ggagtaaata 3480

aattgtctaa ggactaaggt tgtatactat ttaggtgata gatatggggc aaccgtatgg 3540

3600

gagctatagt attctttgct taaataaatt tgattagtgc atgacttctt gaaaacatat 3660

3720

3780

3840

tcatacggtc tgacaacctg tcttatttct atttccccat atgaggaatg ctagttaagt 3900

3960

ctggttccct gaaaatgttt agttagtaat aagtctctta cactatttgt tttgtccaat 4020

aatttatatt ttctgaagac ttaactctag aatacactca tgtcaaaatg aaagaatttc 4080

attgcaaaat attgcttggt acatgacgca tacctgtatt tgttttgtgt cacaacatga 4140

aaaatgatgg gtttcattgg gtaggaaaca catttgaatg gttttacta 4200

agatactaaa atccttggac ttcactctaa ttttagtgcc atttagaact caaggtctca 4260

gtaaaagtag aaataaagcc tgttaacaaa acacaaactg aatattaaaa atgtaactgg 4320

attttcaaag aaatgtttac tggtattacc tgtagatgta tattctttat tatgatcttt 4380

tgtgtaaagt ctggcagaca aatgcaatat ctaattgttg agtccaatat cacaagcagt 4440

acaaaagtat aaaaaagact tggccttttc taatgtgtta aaatacttta tgctggtaat 4500

aacactaaga gtagggcact agaaatttta agtgaagata atgtgttgca gttactgcac 4560

tcaatggctt actattataa accaaaactg ggatcactaa gctccagtca gtcaaaatga 4620

tcaaaattat tgaagagaat aagcaattct gttcttttatt aggacacagt agatacagac 4680

tacaaagtgg agtgtgctta ataagaggta gcatttgtta agtgtcaatt actctattat 4740

cccttggagc ttctcaaaat aaccatataa ggtgtaagat gttaaaggtt atggttacac 4800

tcagtgcaca ggtaagctaa taggctgaga gaagctaaat tacttactgg ggtctcacag 4860

4920

ttacttcatg aatctgtttc tcaattgtgc agaaaaaagg gggctattta taagaaaagc 4980

aataaacaaa caagtaatga tctcaaataa gtaatgcaag aaatagtgag atttcaaaat 5040

cagtggcagc gatttctcag ttctgtccta agtggccttg ctcaatcacc tgctatcttt 5100

5160

agcaaattca caggctcatc tttctaactt gatggtgaat atggaaattc agctaaatgg 5220

5280

tatatgaagg aatataagat aaaggatttt tctaccttca gcaaaaacat acccactaat 5340

tagtaaaatt aataggcgaa aaaaagttgc atgctcttat actgtaatga ttatcatttt 5400

aaaactagct ttttgccttc gagctatcgg ggtaaagacc tacaggaaaa ctactgtcga 5460

aatcctcgag gggaagaagg gggaccctgg tgtttcacaa gcaatccaga ggtacgctac 5520

gaagtctgtg acattcctca gtgttcagaa gttgaatgca tgacctgcaa tggggagagt 5580

tatcgaggtc tcatggatca tacagaatca ggcaagattt gtcagcgctg ggatcatcag 5640

acaccacacc ggcacaaatt cttgcctgaa agatatcccg acaagggctt tgatgataat 5700

tattgccgca atcccgatgg ccagccgagg ccatggtgct atactcttga ccctcacacc 5760

cgctgggagt actgtgcaat taaaacatgc gctgacaata ctatgaatga cactgatgtt 5820

cctttggaaa caactgaatg catccaaggt caaggagaag gctacagggg cactgtcaat 5880

accatttgga atggaattcc atgtcagcgt tgggattctc agtatcctca cgagcatgac 5940

atgactcctg aaaatttcaa gtgcaaggac ctacgagaaa attactgccg aaatccagat 6000

gggtctgaat caccctggtg ttttaccact gatccaaaca tccgagttgg ctactgctcc 6060

caaattccaa actgtgatat gtcacatgga caagattgtt atcgtgggaa tggcaaaaat 6120

6180

atggaagact tacatcgtca tatcttctgg gaaccagatg caagtaagct gaatgagaat 6240

tactgccgaa atccagatga tgatgctcat ggaccctggt gctacacggg aaatccactc 6300

6360

aatttagacc atcccgtaat atcttgtgcc aaaacgaaac aattgcgagt tgtaaatggg 6420

6480

tgcggaggat cattgataaa ggagagttgg gttcttactg cacgacagtg tttcccttct 6540

cgagacttga aagattatga agcttggctt ggaattcatg atgtccacgg aagaggagat 6600

gagaaatgca aacaggttct caatgtttcc cagctggtat atggccctga aggatcagat 6660

ctggttttaa tgaagcttgc caggcctgct gtcctggatg attttgttag tacgattgat 6720

ttacctaatt atggatgcac aattcctgaa aagaccagtt gcagtgttta tggctggggc 6780

tacactggat tgatcaacta tgatggccta ttacgagtgg cacatctcta tataatggga 6840

aatgagaaat gcagccagca tcatcgaggg aaggtgactc tgaatgagtc tgaaatatgt 6900

gctggggctg aaaagattgg atcaggacca tgtgagggggg attatggtgg cccacttgtt 6960

tgtgagcaac ataaaatgag aatggttctt ggtgtcattg ttcctggtcg tggatgtgcc 7020

attccaaatc gtcctggtat ttttgtccga gtagcatatt atgcaaaatg gatacacaaa 7080

attattttaa catataaggt accacagtca tag 7113

<210> 8

<211> 6190

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 8

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgaa ataataacgt aaacttcgtt taaaaggatt cttcttcctg tctttgagaa 780

agtacggcac tgtgcagggg gagaggttga ttgtgaaaaa tcagaggtag atgagaatct 840

tactgagggc tgagggttct ttaaccttgg tggatctcaa cattggttgc acattaaaat 900

cacctgctgc aagcccttga cgaatcttac ttagaagatg acaacacaga acaattaaat 960

cagaatctct ggggagaata gggcaccagt attttttgag ctcccaccat gattccaaag 1020

tgcagccaaa tttgagaacc actgctaaaa gctcaagctt cagattgacc agcttttcca 1080

tctcacctat cgcctaaaga ccaaattgga taaatgtgtt cattacgaca gatgggtact 1140

atttaaagat gagtaaacac aatatactta ggctcgtcag actgagagtt ttaatcatca 1200

ctgaggaaaa acatagatat ctaatactga ctggagtatt agtcaaggct tatttcacac 1260

acaattttat cagaaaccaa agtagtttaa aacagctctc cccttattag taatgcattg 1320

gagggtttac tttaccatgt accttgctga gcactgtacc ttgttaatct catttacttg 1380

taatgagaac cacacagcgg gtagttttat tggttctatt ttacctacat gacaaaactg 1440

aagcataaaa acacttagta agttttcagt gtcatgcaca actaggaagt gacatggcca 1500

gaatataagc ccagtcacca tcactctata acctgcgctt ttaacaactt cagggcatga 1560

cacatttggc cggtcagtag aacccatgct gtgatttgtt tttgcagtgg tggtgatgac 1620

1680

ttcttcatta ggaaacagag atgagtttatt gaccaacaca gaaagaaaaa gagtttgttg 1740

ctccacactg ggattaaacc tatgatcttg gcctaattaa cactagctag taagtgtcca 1800

agctgatcat ctctacaaca tttcaataac agaaaacaac aattttcaaa attagttact 1860

tacaattatg tagaaatgcc tctaaaacac agtattttcc ttatattaca aaaacaaaaa 1920

tttaattgg ttttgtcctc ttttgagagt ttgcatggtg ttactccctg catagtgaag 1980

aaaacatttt atttaagtag atggatctaa gtttttcatg aacaaaggaa tgacatttga 2040

aatcaatcct accctagtcc aggagaatgc attagattaa cctagtagag gtcttatttc 2100

accctgagtt ttctatgatc gtgattctct gctggaggag taattgtgaa atagatctct 2160

ctgggaactg gcttcctagt ccaatcagct cttttaccaa tgaacacttc cttgtgatat 2220

agatgtttat ggccgagagg atctcttcct ttctacctgt atttgtccta ataaattgtt 2280

2340

gtatatgggt gtatcacttt gtgtatttcg gtgtgcatgt gtagagggga caaaaatcct 2400

2460

cttaaataat cttaatatat aaaatgatat agaaaaaggg aaattatagt tcgtattatt 2520

catctaagtg aagagattaa aacccaggga gtaaataaat tgtctaagga ctaaggttgt 2580

atactattta ggtgatagat atggggcaac cgtatgggtt ttatgattaa caaataaact 2640

tctcaccact ctaccatatc aacttttcca taaaagagag ctatagtatt ctttgcttaa 2700

ataaatttga ttagtgcatg acttcttgaa aacatataaa gcaaaagtca catttgattc 2760

tatcagaaaa gtgagtaagc catggcccaa acaaaagatg cattaaaata ttctggaatg 2820

atggagctaa aagtaagaaa aatgactttt taaaaaagtt tactgttagg aattgtgaaa 2880

ttatgctgaa ttttagttgc attataattt ttgtcagtca tacggtctga caacctgtct 2940

tatttctatt tccccatatg aggaatgcta gttaagtatg gatattaact attactactt 3000

agatgcattg aagttgcata atatggataa tacttcactg gttccctgaa aatgtttagt 3060

tagtaataag tctcttacac tatttgtttt gtccaataat ttatattttc tgaagactta 3120

actctagaat acactcatgt caaaatgaaa gaatttcatt gcaaaatatt gcttggtaca 3180

3240

3300

actctaattt tagtgccatt tagaactcaa ggtctcagta aaagtagaaa taaagcctgt 3360

3420

tattacctgt agatgtatat tctttattat gatcttttgt gtaaagtctg gcagacaaat 3480

gcaatatcta attgttgagt ccaatatcac aagcagtaca aaagtataaa aaagacttgg 3540

ccttttctaa tgtgttaaaa tactttatgc tggtaataac actaaagta gggcactaga 3600

aattttaagt gaagataatg tgttgcagtt actgcactca atggcttact attataaacc 3660

aaaactggga tcactaagct ccagtcagtc aaaatgatca aaattattga agagaataag 3720

caattctgtt ctttattagg acacagtaga tacagactac aaagtggagt gtgcttaata 3780

agaggtagca tttgttaagt gtcaattact ctattatccc ttggagcttc tcaaaataac 3840

catataaggt gtaagatgtt aaaggttatg gttacactca gtgcacaggt aagctaatag 3900

3960

gccgattt aactggattc tgggctcttt attcatgtta cttcatgaat ctgtttctca 4020

attgtgcaga aaaaaggggg ctatttataa gaaaagcaat aaacaaacaa gtaatgatct 4080

caaataagta atgcaagaaa tagtgagatt tcaaaatcag tggcagcgat ttctcagttc 4140

tgtcctaagt ggccttgctc aatcacctgc tatcttttag tggagctttg aaattatgtt 4200

tcagacaact tcgattcagt tctagaatgt ttgactcagc aaattcacag gctcatcttt 4260

ctaacttgat ggtgaatatg gaaattcagc taaatggatg ttaataaaat tcaaacgttt 4320

taaggacaga tggaaatgac agaattttaa ggtaaaatat atgaaggaat ataagataaa 4380

ggatttttct accttcagca aaaacatacc cactaattag taaaattaat aggcgaaaaa 4440

aagttgcatg ctcttatact gtaatgatta tcattttaaa actagctttt tgccttcgag 4500

ctatcggggt aaagacctac aggaaaacta ctgtcgaaat cctcgagggg aagaaggggg 4560

accctggtgt ttcacaagca atccagaggt acgctacgaa gtctgtgaca ttcctcagtg 4620

ttcagaagtt gaatgcatga cctgcaatgg ggagagttat cgaggtctca tggatcatac 4680

agaatcaggc aagatttgtc agcgctggga tcatcagaca ccacaccggc acaaattctt 4740

gcctgaaaga tatcccgaca agggctttga tgataattat tgccgcaatc ccgatggcca 4800

gccgaggcca tggtgctata ctcttgaccc tcacacccgc tgggagtact gtgcaattaa 4860

aacatgcgct gacaatacta tgaatgacac tgatgttcct ttggaaacaa ctgaatgcat 4920

ccaaggtcaa ggagaaggct acaggggcac tgtcaatacc atttggaatg gaattccatg 4980

tcagcgttgg gattctcagt atcctcacga gcatgacatg actcctgaaa atttcaagtg 5040

caaggaccta cgagaaaatt actgccgaaa tccagatggg tctgaatcac cctggtgttt 5100

taccactgat ccaaacatcc gagttggcta ctgctcccaa attccaaact gtgatatgtc 5160

acatggacaa gattgttatc gtgggaatgg caaaaattat atgggcaact tatcccaaac 5220

aagatctgga ctaacatgtt caatgtggga caagaacatg gaagacttac atcgtcatat 5280

cttctgggaa ccagatgcaa gtaagctgaa tgagaattac tgccgaaatc cagatgatga 5340

tgctcatgga ccctggtgct acacgggaaa tccactcatt ccttgggatt attgccctat 5400

ttctcgttgt gaaggtgata ccacacctac aatagtcaat ttagaccatc ccgtaatatc 5460

ttgtgccaaa acgaaacaat tgcgagttgt aaatgggatt ccaacacgaa caaacatagg 5520

atggatggtt agtttgagat acagaaataa acatatctgc ggaggatcat tgataaagga 5580

gagttgggtt cttactgcac gacagtgttt cccttctcga gacttgaaag attatgaagc 5640

ttggcttgga attcatgatg tccacggaag aggagatgag aaatgcaaac aggttctcaa 5700

tgtttcccag ctggtatatg gccctgaagg atcagatctg gttttaatga agcttgccag 5760

gcctgctgtc ctggatgatt ttgttagtac gattgattta cctaattatg gatgcacaat 5820

tcctgaaaag accagttgca gtgtttatgg ctggggctac actggattga tcaactatga 5880

tggcctatta cgagtggcac atctctatat aatgggaaat gagaaatgca gccagcatca 5940

tcgagggaag gtgactctga atgagtctga aatatgtgct ggggctgaaa agattggatc 6000

aggaccatgt gagggggatt atggtggccc acttgtttgt gagcaacata aaatgagaat 6060

ggttcttggt gtcattgttc ctggtcgtgg atgtgccatt ccaaatcgtc ctggtatttt 6120

6180

acagtcatag 6190

<210> 9

<211> 5190

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 9

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgaa ataataacgt aaacttcgtt taaaaggatt cttcttcctg tctttgagaa 780

agtacggcac tgtgcagggg gagaggttga ttgtgaaaaa tcagaggtag atgagaatct 840

tactgagggc tgagggttct ttaaccttgg tggatctcaa cattggttgc acattaaaat 900

cacctgctgc aagcccttga cgaatcttac ttagaagatg acaacacaga acaattaaat 960

cagaatctct ggggagaata gggcaccagt attttttgag ctcccaccat gattccaaag 1020

tgcagccaaa tttgagaacc actgctaaaa gctcaagctt cagattgacc agcttttcca 1080

tctcacctat cgcctaaaga ccaaattgga taaatgtgtt cattacgaca gatgggtact 1140

atttaaagat gagtaaacac aatatactta ggctcgtcag actgagagtt ttaatcatca 1200

ctgaggaaaa acatagatat ctaatactga ctggagtatt agtcaaggct tatttcacac 1260

acaattttat cagaaaccaa agtagtttaa aacagctctc cccttattag taatgcattg 1320

gagggtttac tttaccatgt accttgctga gcactgtacc ttgttaatct catttacttg 1380

taatgagaac cacacagcgg gtagttttat tggttctatt ttacctacat gacaaaactg 1440

aagcataaaa acacttagta agttttcagt gtcatgcaca actaggaagt gacatggcca 1500

gaatataagc ccagtcacca tcactctata acctgcgctt ttaacaactt cagggcatga 1560

cacatttggc cggtcagtag aacccatgct gtgatttgtt tttgcagtgg tggtgatgac 1620

1680

ttcttcatta ggaaacagag atgagtttatt gaccaacaca gaaagaaaaa gagtttgttg 1740

ctccacactg ggattaaacc tatgatcttg gcctaattaa cactagctag taagtgtcca 1800

agctgatcat ctctacaaca tttcaataac agaaaacaac aattttcaaa attagttact 1860

tacaattatg tagaaatgcc tctaaaacac agtattttcc ttatattaca aaaacaaaaa 1920

tttaattgg ttttgtcctc ttttgagagt ttgcatggtg ttactccctg catagtgaag 1980

aaaacatttt atttaagtag atggatctaa gtttttcatg aacaaaggaa tgacatttga 2040

aatcaatcct accctagtcc aggagaatgc attagattaa cctagtagag gtcttatttc 2100

accctgagtt ttctatgatc gtgattctct gctggaggag taattgtgaa atagatctct 2160

ctgggaactg gcttcctagt ccaatcagct cttttaccaa tgaacacttc cttgtgatat 2220

2280

tactaaaatc cttggacttc actctaattt tagtgccatt tagaactcaa ggtctcagta 2340

2400

2460

gtaaagtctg gcagacaaat gcaatatcta attgttgagt ccaatatcac aagcagtaca 2520

aaagtataaa aaagacttgg ccttttctaa tgtgttaaaa tactttatgc tggtaataac 2580

actaagagta gggcactaga aattttaagt gaagataatg tgttgcagtt actgcactca 2640

atggcttact attataaacc aaaactggga tcactaagct ccagtcagtc aaaatgatca 2700

aaattattga agagaataag caattctgtt ctttattagg acacagtaga tacagactac 2760

aaagtggagt gtgcttaata agaggtagca tttgttaagt gtcaattact ctattatccc 2820

ttggagcttc tcaaaataac catataaggt gtaagatgtt aaaggttatg gttacactca 2880

gtgcacaggt aagctaatag gctgagagaa gctaaattac ttactggggt ctcacagtaa 2940

gaaagtgagc tgaagtttca gcccagattt aactggattc tgggctcttt attcatgtta 3000

cttcatgaat ctgtttctca attgtgcaga aaaaaggggg ctatttataa gaaaagcaat 3060

3120

tggcagcgat ttctcagttc tgtcctaagt ggccttgctc aatcacctgc tatcttttag 3180

tggagctttg aaattatgtt tcagacaact tcgattcagt tctagaatgt ttgactcagc 3240

aaattcacag gctcatcttt ctaacttgat ggtgaatatg gaaattcagc taaatggatg 3300

3360

atgaaggaat ataagataaa ggatttttct accttcagca aaaacatacc cactaattag 3420

taaaattaat aggcgaaaaa aagttgcatg ctcttatact gtaatgatta tcattttaaa 3480

actagctttt tgccttcgag ctatcggggt aaagacctac aggaaaacta ctgtcgaaat 3540

cctcgagggg aagaaggggg accctggtgt ttcacaagca atccagaggt acgctacgaa 3600

3660

cgaggtctca tggatcatac agaatcaggc aagatttgtc agcgctggga tcatcagaca 3720

ccacaccggc acaaattctt gcctgaaaga tatcccgaca agggctttga tgataattat 3780

tgccgcaatc ccgatggcca gccgaggcca tggtgctata ctcttgaccc tcacacccgc 3840

tgggagtact gtgcaattaa aacatgcgct gacaatacta tgaatgacac tgatgttcct 3900

ttggaaacaa ctgaatgcat ccaaggtcaa ggagaaggct acaggggcac tgtcaatacc 3960

atttggaatg gaattccatg tcagcgttgg gattctcagt atcctcacga gcatgacatg 4020

actcctgaaa atttcaagtg caaggaccta cgagaaaatt actgccgaaa tccagatggg 4080

tctgaatcac cctggtgttt taccactgat ccaaacatcc gagttggcta ctgctcccaa 4140

attccaaact gtgatatgtc acatggacaa gattgttatc gtgggaatgg caaaaattat 4200

atgggcaact tatcccaaac aagatctgga ctaacatgtt caatgtggga caagaacatg 4260

gaagacttac atcgtcatat cttctgggaa ccagatgcaa gtaagctgaa tgagaattac 4320

tgccgaaatc cagatgatga tgctcatgga ccctggtgct acacgggaaa tccactcatt 4380

ccttgggatt attgccctat ttctcgttgt gaaggtgata ccacacctac aatagtcaat 4440

ttagaccatc ccgtaatatc ttgtgccaaa acgaaacaat tgcgagttgt aaatgggatt 4500

ccaacacgaa caaacatagg atggatggtt agtttgagat acagaaataa acatatctgc 4560

ggaggatcat tgataaagga gagttgggtt cttactgcac gacagtgttt cccttctcga 4620

4680

aaatgcaaac aggttctcaa tgtttcccag ctggtatatg gccctgaagg atcagatctg 4740

gttttaatga agcttgccag gcctgctgtc ctggatgatt ttgttagtac gattgattta 4800

cctaattatg gatgcacaat tcctgaaaag accagttgca gtgtttatgg ctggggctac 4860

actggattga tcaactatga tggcctatta cgagtggcac atctctatat aatgggaaat 4920

gagaaatgca gccagcatca tcgagggaag gtgactctga atgagtctga aatatgtgct 4980

ggggctgaaa agattggatc aggaccatgt gagggggatt atggtggccc acttgtttgt 5040

gagcaacata aaatgagaat ggttcttggt gtcattgttc ctggtcgtgg atgtgccatt 5100

ccaaatcgtc ctggtatttt tgtccgagta gcatattatg caaaatggat acacaaaatt 5160

attttaacat ataaggtacc acagtcatag 5190

<210> 10

<211> 4241

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 10

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgaa ataataacgt aaacttcgtt taaaaggatt cttcttcctg tctttgagaa 780

agtacggcac tgtgcagggg gagaggttga ttgtgaaaaa tcagaggtag atgagaatct 840

tactgagggc tgagggttct ttaaccttgg tggatctcaa cattggttgc acattaaaat 900

cacctgctgc aagcccttga cgaatcttac ttagaagatg acaacacaga acaattaaat 960

cagaatctct ggggagaata gggcaccagt attttttgag ctcccaccat gattccaaag 1020

tgcagccaaa tttgagaacc actgctaaaa gctcaagctt cagattgacc agcttttcca 1080

tctcacctat cgcctaaaga ccaaattgga taaatgtgtt cattacgaca gatgggtact 1140

atttaaagat gagtaaacac aatatactta ggctcgtcag actgagagtt ttaatcatca 1200

ctgaggaaaa acatagatat ctaatactga ctggagtatt agtcaaggct tatttcacac 1260

acaattttat cagaaaccaa agtagtttaa aacagctctc cccttattag taatgcattg 1320

gagggtttac tttaccatgt accttgctga gcactgtacc ttgttaatct catttacttg 1380

taatgagaac cacacagcgg gtagttttat tggttctatt ttacctacat gacaaaactg 1440

aagcataaaa acacttagta agttttcagt gtcatgcaca actaggaagt gacatggcca 1500

gaatataagc ccagtcacca tcactctata acctgcgctt ttaacaactt cagggcatga 1560

cacatttggc cggtcagtag aacccatgct gtgatttgtt tttgcagtgg tggtgatgac 1620

1680

ttcttcatta ggaaacagag atgagtttatt gaccaacaca gaaagaaaaa gagtttgttg 1740

ctccacactg ggattaaacc tatgatcttg gcctaattaa cactagctag taagtgtcca 1800

agctgatcat ctctacaaca tttcaataac agaaaacaac aattttcaaa attagttact 1860

tacaattatg tagaaatgcc tctaaaacac agtattttcc ttatattaca aaaacaaaaa 1920

tttaattgg ttttgtcctc ttttgagagt ttgcatggtg ttactccctg catagtgaag 1980

aaaacatttt atttaagtag atggatctaa gtttttcatg aacaaaggaa tgacatttga 2040

aatcaatcct accctagtcc aggagaatgc attagattaa cctagtagag gtcttatttc 2100

accctgagtt ttctatgatc gtgattctct gctggaggag taattgtgaa atagatctct 2160

ctgggaactg gcttcctagt ccaatcagct cttttaccaa tgaacacttc cttgtgatat 2220

agatgtttat ggccgagagg atccttatgt ttcagacaac ttcgattcag ttctagaatg 2280

tttgactcag caaattcaca ggctcatctt tctaacttga tggtgaatat ggaaattcag 2340

ctaaatggat gttaataaaa ttcaaacgtt ttaaggacag atggaaatga cagaatttta 2400

aggtaaaata tatgaaggaa tataagataa aggatttttc taccttcagc aaaaacatac 2460

ccactaatta gtaaaattaa taggcgaaaa aaagttgcat gctcttatac tgtaatgatt 2520

atcattttaa aactagcttt ttgccttcga gctatcgggg taaagaccta caggaaaact 2580

actgtcgaaa tcctcgaggg gaagaagggg gaccctggtg tttcacaagc aatccagagg 2640

tacgctacga agtctgtgac attcctcagt gttcagaagt tgaatgcatg acctgcaatg 2700

gggagagtta tcgaggtctc atggatcata cagaatcagg caagatttgt cagcgctggg 2760

atcatcagac accacaccgg cacaaattct tgcctgaaag atatcccgac aagggctttg 2820

atgataatta ttgccgcaat cccgatggcc agccgaggcc atggtgctat actcttgacc 2880

ctcacacccg ctgggagtac tgtgcaatta aaacatgcgc tgacaatact atgaatgaca 2940

ctgatgttcc tttggaaaca actgaatgca tccaaggtca aggagaaggc tacaggggca 3000

ctgtcaatac catttggaat ggaattccat gtcagcgttg ggattctcag tatcctcacg 3060

agcatgacat gactcctgaa aatttcaagt gcaaggacct acgagaaaat tactgccgaa 3120

atccagatgg gtctgaatca ccctggtgtt ttaccactga tccaaacatc cgagttggct 3180

actgctccca aattccaaac tgtgatatgt cacatggaca agattgttat cgtgggaatg 3240

gcaaaaatta tatgggcaac ttatcccaaa caagatctgg actaacatgt tcaatgtggg 3300

acaagaacat ggaagactta catcgtcata tcttctggga accagatgca agtaagctga 3360

atgagaatta ctgccgaaat ccagatgatg atgctcatgg accctggtgc tacacgggaa 3420

atccactcat tccttgggat tattgcccta tttctcgttg tgaaggtgat accacaccta 3480

aacgaaacaa ttgcgagttg 3540

3600

3660

tcccttctcg agacttgaaa gattatgaag cttggcttgg aattcatgat gtccacggaa 3720

gaggagatga gaaatgcaaa caggttctca atgtttccca gctggtatat ggccctgaag 3780

gatcagatct ggttttaatg aagcttgcca ggcctgctgt cctggatgat tttgttagta 3840

cgattgattt acctaattat ggatgcacaa ttcctgaaaa gaccagttgc agtgtttatg 3900

gctggggcta cactggattg atcaactatg atggcctatt acgagtggca catctctata 3960

taatgggaaa tgagaaatgc agccagcatc atcgagggaa ggtgactctg aatgagtctg 4020

aaatatgtgc tggggctgaa aagattggat caggaccatg tgagggggat tatggtggcc 4080

cacttgtttg tgagcaacat aaaatgagaa tggttcttgg tgtcattgtt cctggtcgtg 4140

gatgtgccat tccaaatcgt cctggtattt ttgtccgagt agcatattat gcaaaatgga 4200

tacacaaaat tattttaaca tataaggtac cacagtcata g 4241

<210> 11

<211> 5602

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 11

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgga tccagtatat taataaaatc cctttttgta ttcaatgagg gaaacacata 780

tctgcatgat ggtttaacac ttttaagtgt 840

tgactaaaga ttaattttac agaaaataga aaaagaaata tgtttctgtc tggaggaatg 900

atttattgtt gacccctaaa ttgaaatatt ttactagtgg cttaatggaa agatgatgaa 960

agatgatgaa attaatgtag aagcttaact agaaaatcag gtgacctgat atctacatct 1020

gtatccttca ttggccaccc agcattcatt aatgaatcag atgatggaat agatcaagtt 1080

tcctaggaac acagtgaata ttaaaagaaa acaaagggag cctagcacct agaagaccta 1140

gtttatttt caaagtatat ttggatgtaa cccaatttta aacatttcct cacttgtctc 1200

tcttaaagcc ttgccaacag caaggacaga gaaccaaaaa tagtgtatat atgaataaat 1260

gcttattaca gaatctgctg actggcacat gctttgtgtg taatggggttc tcataaacac 1320

ttgttgaatg aacacacata agtgaaagag catggctagg cttcatccct tggtcaaata 1380

tggggtgcta aagaaaagca ggggaaatac attgggacac taacaaaaaa aaacagttaa 1440

tttaggtaaa agataaaata caccacagaa tgaagaaaag agatgaccca gactgctctt 1500

taaccttcat gtcctagaga ggtttttgat atgaattgca ttcagaattg tggaaaggag 1560

cccatctttt ctcttcattt tgattttatt aactccaatg ggggaatttt attcgtgttt 1620

tggccatatc tacttttgat ttctacatta ttctctcttc ctttctacct gtatttgtcc 1680

taataaattg ttgacttatt aattcactac ttcctcacag cttttttttg gctttacaaa 1740

tccactggaa aggtatatgg gtgtatcact ttgtgtattt cggtgtgcat gtgtagaggg 1800

gacaaaaatc ctctctcaaa ctataaatat tgagtatttg tgtattgaac atttgctata 1860

actactaggt ttcttaaata atcttaatat ataaaatgat atagaaaaag ggaaattata 1920

gttcgtatta ttcatctaag tgaagagatt aaaacccagg gagtaaataa attgtctaag 1980

gactaaggtt gtatactatt taggtgatag atatggggca accgtatggg ttttatgatt 2040

aacaaataaa cttctcacca ctctaccata tcaacttttc cataaaagag agctatagta 2100

ttctttgctt aaataaattt gattagtgca tgacttcttg aaaacatata aagcaaaagt 2160

cacatttgat tctatcagaa aagtgagtaa gccatggccc aaacaaaaga tgcattaaaa 2220

tattctggaa tgatggagct aaaagtaaga aaaatgactt tttaaaaaag tttactgtta 2280

ggaattgtga aattatgctg aattttagtt gcattataat ttttgtcagt catacggtct 2340

gacaacctgt cttatttcta tttccccata tgaggaatgc tagttaagta tggatattaa 2400

ctattactac ttagatgcat tgaagttgca taatatggat aatacttcac tggttccctg 2460

aaaatgttta gttagtaata agtctcttac actatttgtt ttgtccaata atttatattt 2520

tctgaagact taactctaga atacactcat gtcaaaatga aagaatttca ttgcaaaata 2580

ttgcttggta catgacgcat acctgtattt gttttgtgtc acaacatgaa aaatgatggt 2640

ttattagaag ttttcattgggg taggaaacac atttgaatgg tatttactaa gatactaaaa 2700

tccttggact tcactctaat tttagtgcca tttagaactc aaggtctcag taaaagtaga 2760

aataaagcct gttaacaaaa cacaaactga atattaaaaa tgtaactgga ttttcaaaga 2820

2880

tggcagacaa atgcaatatc taattgttga gtccaatatc acaagcagta caaaagtata 2940

aaaaagactt ggccttttct aatgtgttaa aatactttat gctggtaata acactaagag 3000

tagggcacta gaaattttaa gtgaagataa tgtgttgcag ttactgcact caatggctta 3060

ctattataaa ccaaaactgg gatcactaag ctccagtcag tcaaaatgat caaaattatt 3120

gaagagaata agcaattctg ttctttatta ggacacagta gatacagact acaaagtgga 3180

gtgtgcttaa taagaggtag catttgttaa gtgtcaatta ctctattatc ccttggagct 3240

tctcaaaata accatataag gtgtaagatg ttaaaggtta tggttacact cagtgcacag 3300

gtaagctaat aggctgagag aagctaaatt acttactggg gtctcacagt aagaaagtga 3360

3420

3480

3540

atttctcagt tctgtcctaa gtggccttgc tcaatcacct gctatctttt agtggagctt 3600

3660

aggctcatct ttctaacttg atggtgaata tggaaattca gctaaatgga tgttaataaa 3720

3780

3840

ataggcgaaa aaaagttgca tgctcttata ctgtaatgat tatcatttta aaactagctt 3900

tttgccttcg agctatcgg gtaaagacct acaggaaaac tactgtcgaa atcctcgagg 3960

ggaagaaggg ggaccctggt gtttcacaag caatccagag gtacgctacg aagtctgtga 4020

cattcctcag tgttcagaag ttgaatgcat gacctgcaat ggggagagtt atcgaggtct 4080

catggatcat acagaatcag gcaagatttg tcagcgctgg gatcatcaga caccacaccg 4140

gcacaaattc ttgcctgaaa gatatcccga caagggcttt gatgataatt attgccgcaa 4200

tcccgatggc cagccgaggc catggtgcta tactcttgac cctcacaccc gctgggagta 4260

ctgtgcaatt aaaacatgcg ctgacaatac tatgaatgac actgatgttc ctttggaaac 4320

aactgaatgc atccaaggtc aaggagaagg ctacaggggc actgtcaata ccatttggaa 4380

tggaattcca tgtcagcgtt gggattctca gtatcctcac gagcatgaca tgactcctga 4440

aaatttcaag tgcaaggacc tacgagaaaa ttactgccga aatccagatg ggtctgaatc 4500

accctggtgt tttaccactg atccaaacat ccgagttggc tactgctccc aaattccaaa 4560

ctgtgatatg tcacatggac aagattgtta tcgtgggaat ggcaaaaatt atatgggcaa 4620

cttatcccaa acaagatctg gactaacatg ttcaatgtgg gacaagaaca tggaagactt 4680

acatcgtcat atcttctggg aaccagatgc aagtaagctg aatgagaatt actgccgaaa 4740

tccagatgat gatgctcatg gaccctggtg ctacacggga aatccactca ttccttggga 4800

ttattgccct atttctcgtt gtgaaggtga taccacacct acaatagtca atttagacca 4860

tcccgtaata tcttgtgcca aaacgaaaca attgcgagtt gtaaatggga ttccaacacg 4920

aacaaacata ggatggatgg ttagtttgag atacagaaat aaacatatct gcggaggatc 4980

attgataaag gagagttggg ttcttactgc acgacagtgt ttcccttctc gagacttgaa 5040

agattatgaa gcttggcttg gaattcatga tgtccacgga agaggagatg agaaatgcaa 5100

acaggttctc aatgtttccc agctggtata tggccctgaa ggatcagatc tggttttaat 5160

gaagcttgcc aggcctgctg tcctggatga ttttgttagt acgattgatt tacctaatta 5220

tggatgcaca attcctgaaa agaccagttg cagtgtttat ggctggggct acactggatt 5280

gatcaactat gatggcctat tacgagtggc acatctctat ataatgggaa atgagaaatg 5340

cagccagcat catcgaggga aggtgactct gaatgagtct gaaatatgtg ctggggctga 5400

aaagattgga tcaggaccat gtgaggggga ttatggtggc ccacttgttt gtgagcaaca 5460

taaaatgaga atggttcttg gtgtcattgt tcctggtcgt ggatgtgcca ttccaaatcg 5520

5580

atataaggta ccacagtcat ag 5602

<210> 12

<211> 4679

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 12

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgga tctcttcctt tctacctgta tttgtcctaa taaattgttg acttattaat 780

tcactacttc ctcacagctt ttttttggct ttacaaatcc actggaaagg tatatgggtg 840

tatcactttg tgtatttcgg tgtgcatgtg tagaggggac aaaaatcctc tctcaaacta 900

taaatattga gtatttgtgt attgaacatt tgctataact actaggtttc ttaaataatc 960

ttaatatata aaatgatata gaaaaaggga aattatagtt cgtattattc atctaagtga 1020

aggattaaa acccagggag taaataaatt gtctaaggac taaggttgta tactatttag 1080

gtgatagata tggggcaacc gtatgggttt tatgattaac aaataaactt ctcaccactc 1140

taccatatca acttttccat aaaagagagc tatagtattc tttgcttaaa taaatttgat 1200

tagtgcatga cttcttgaaa acatataaag caaaagtcac atttgattct atcagaaaag 1260

tgagtaagcc atggcccaaa caaaagatgc attaaaatat tctggaatga tggagctaaa 1320

1380

tttagttgca ttataatttt tgtcagtcat acggtctgac aacctgtctt atttctattt 1440

ccccatatga ggaatgctag ttaagtatgg atattaacta ttactactta gatgcattga 1500

agttgcataa tatggataat acttcactgg ttccctgaaa atgtttagtt agtaataagt 1560

ctcttacact atttgttttg tccaataatt tatattttct gaagacttaa ctctagaata 1620

cactcatgtc aaaatgaaag aatttcattg caaaatattg cttggtacat gacgcatacc 1680

tgtatttgtt ttgtgtcaca acatgaaaaa tgatggttta ttagaagttt cattgggtag 1740

gaaacacatt tgaatggtat ttactaagat actaaaatcc ttggacttca ctctaatttt 1800

agtgccattt agaactcaag gtctcagtaa aagtagaaat aaagcctgtt aacaaaacac 1860

aaactgaata ttaaaaatgt aactggattt tcaaagaaat gtttactggt attacctgta 1920

gatgtatatt ctttattatg atcttttgtg taaagtctgg cagacaaatg caatatctaa 1980

ttgttgagtc caatatcaca agcagtacaa aagtataaaa aagacttggc cttttctaat 2040

gtgttaaaat actttatgct ggtaataaca ctaagagtag ggcactagaa attttaagtg 2100

aagataatgt gttgcagtta ctgcactcaa tggcttacta ttataaacca aaactgggat 2160

cactaagctc cagtcagtca aaatgatcaa aattattgaa gagaataagc aattctgttc 2220

tttattagga cacagtagat acagactaca aagtggagtg tgcttaataa gaggtagcat 2280

ttgttaagtg tcaattactc tattatccct tggagcttct caaaataacc atataaggtg 2340

taagatgtta aaggttatgg ttacactcag tgcacaggta agctaatagg ctgagagaag 2400

ctaaattact tactggggtc tcacagtaag aaagtgagct gaagtttcag cccagattta 2460

actggattct gggctcttta ttcatgttac ttcatgaatc tgtttctcaa ttgtgcagaa 2520

2580

tgcaagaaat agtgagattt caaaatcagt ggcagcgatt tctcagttct gtcctaagtg 2640

gccttgctca atcacctgct atcttttagt ggagctttga aattatgttt cagacaactt 2700

cgattcagtt ctagaatgtt tgactcagca aattcacagg ctcatctttc taacttgatg 2760

gtgaatatgg aaattcagct aaatggatgt taataaaatt caaacgtttt aaggacagat 2820

ggaaatgaca gaattttaag gtaaaatata tgaaggaata taagataaag gatttttcta 2880

ccttcagcaa aaacataccc actaattagt aaaattaata ggcgaaaaaa agttgcatgc 2940

tcttatactg taatgattat cattttaaaa ctagcttttt gccttcgagc tatcggggta 3000

aagacctaca ggaaaactac tgtcgaaatc ctcgagggga agaaggggga ccctggtgtt 3060

tcacaagcaa tccagaggta cgctacgaag tctgtgacat tcctcagtgt tcagaagttg 3120

aatgcatgac ctgcaatggg gagagttatc gaggtctcat ggatcataca gaatcaggca 3180

agatttgtca gcgctgggat catcagacac cacaccggca caaattcttg cctgaaagat 3240

atcccgacaa gggctttgat gataattatt gccgcaatcc cgatggccag ccgaggccat 3300

ggtgctatac tcttgaccct cacacccgct gggagtactg tgcaattaaa acatgcgctg 3360

acaatactat gaatgacact gatgttcctt tggaaacaac tgaatgcatc caaggtcaag 3420

gagaaggcta caggggcact gtcaatacca tttggaatgg aattccatgt cagcgttggg 3480

3540

gagaaaatta ctgccgaaat ccagatgggt ctgaatcacc ctggtgtttt accactgatc 3600

3660

3720

taacatgttc aatgtgggac aagaacatgg aagacttaca tcgtcatatc ttctgggaac 3780

cagatgcaag taagctgaat gagaattact gccgaaatcc agatgatgat gctcatggac 3840

cctggtgcta cacgggaaat ccactcattc cttgggatta ttgccctatt tctcgttgtg 3900

aaggtgatac cacacctaca atagtcaatt tagaccatcc cgtaatatct tgtgccaaaa 3960

cgaaacaatt gcgagttgta aatgggattc caacacgaac aaacatagga tggatggtta 4020

gtttgagata cagaaataaa catatctgcg gaggatcatt gataaaggag agttgggttc 4080

ttactgcacg acagtgtttc ccttctcgag acttgaaaga ttatgaagct tggcttggaa 4140

ttcatgatgt ccacggaaga ggagatgaga aatgcaaaca ggttctcaat gtttcccagc 4200

tggtatatgg ccctgaagga tcagatctgg ttttaatgaa gcttgccagg cctgctgtcc 4260

tggatgattt tgttagtacg attgatttac ctaattatgg atgcacaatt cctgaaaaga 4320

ccagttgcag tgtttatggc tggggctaca ctggattgat caactatgat ggcctattac 4380

gagtggcaca tctctatata atgggaaatg agaaatgcag ccagcatcat cgagggaagg 4440

tgactctgaa tgagtctgaa atatgtgctg gggctgaaaa gattggatca ggaccatgtg 4500

agggggatta tggtggccca cttgtttgtg agcaacataa aatgagaatg gttcttggtg 4560

tcattgttcc tggtcgtgga tgtgccattc caaatcgtcc tggtattttt gtccgagtag 4620

catattatgc aaaatggata cacaaaatta ttttaacata taaggtacca cagtcatag 4679

<210> 13

<211> 3679

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 13

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgga tcctgggtag gaaacacatt tgaatggtat ttactaagat actaaaatcc 780

ttggacttca ctctaatttt agtgccattt agaactcaag gtctcagtaa aagtagaaat 840

aaagcctgtt aacaaaacac aaactgaata ttaaaaatgt aactggattt tcaaagaaat 900

960

1020

aagacttggc cttttctaat gtgttaaaat actttatgct ggtaataaca ctaaagtag 1080

ggcactagaa attttaagtg aagataatgt gttgcagtta ctgcactcaa tggcttacta 1140

ttataaacca aaactgggat cactaagctc cagtcagtca aaatgatcaa aattattgaa 1200

1260

tgcttaataa gaggtagcat ttgttaagtg tcaattactc tattatccct tggagcttct 1320

caaaataacc atataaggtg taagatgtta aaggttatgg ttacactcag tgcacaggta 1380

agctaatagg ctgagagaag ctaaattact tactggggtc tcacagtaag aaagtgagct 1440

gaagtttcag cccagattta actggattct gggctcttta ttcatgttac ttcatgaatc 1500

tgtttctcaa ttgtgcagaa aaaagggggc tatttataag aaaagcaata aacaaacaag 1560

taatgatctc aaataagtaa tgcaagaaat agtgagattt caaaatcagt ggcagcgatt 1620

tctcagttct gtcctaagtg gccttgctca atcacctgct atcttttagt ggagctttga 1680

aattatgttt cagacaactt cgattcagtt ctagaatgtt tgactcagca aattcacagg 1740

ctcatctttc taacttgatg gtgaatatgg aaattcagct aaatggatgt taataaaatt 1800

caaacgtttt aaggacagat ggaaatgaca gaattttaag gtaaaatata tgaaggaata 1860

taagataaag gatttttcta ccttcagcaa aaacataccc actaattagt aaaattaata 1920

ggcgaaaaaa agttgcatgc tcttatactg taatgattat cattttaaaa ctagcttttt 1980

gccttcgagc tatcggggta aagacctaca ggaaaactac tgtcgaaatc ctcgagggga 2040

agaaggggga ccctggtgtt tcacaagcaa tccagaggta cgctacgaag tctgtgacat 2100

tcctcagtgt tcagaagttg aatgcatgac ctgcaatggg gagagttatc gaggtctcat 2160

ggatcataca gaatcaggca agatttgtca gcgctgggat catcagacac cacaccggca 2220

caaattcttg cctgaaagat atcccgacaa gggctttgat gataattatt gccgcaatcc 2280

cgatggccag ccgaggccat ggtgctatac tcttgaccct cacacccgct gggagtactg 2340

tgcaattaaa acatgcgctg acaatactat gaatgacact gatgttcctt tggaaacaac 2400

tgaatgcatc caaggtcaag gagaaggcta caggggcact gtcaatacca tttggaatgg 2460

aattccatgt cagcgttggg attctcagta tcctcacgag catgacatga ctcctgaaaa 2520

tttcaagtgc aaggacctac gagaaaatta ctgccgaaat cggatggggt ctgaatcacc 2580

ctggtgtttt accactgatc caaacatccg agttggctac tgctcccaaa ttccaaactg 2640

tgatatgtca catggacaag attgttatcg tgggaatggc aaaaattata tgggcaactt 2700

atcccaaaca agatctggac taacatgttc aatgtgggac aagaacatgg aagacttaca 2760

tcgtcatatc ttctgggaac cagatgcaag taagctgaat gagaattact gccgaaatcc 2820

agatgatgat gctcatggac cctggtgcta cacgggaaat ccactcattc cttgggatta 2880

ttgccctatt tctcgttgtg aaggtgatac cacacctaca atagtcaatt tagaccatcc 2940

cgtaatatct tgtgccaaaa cgaaacaatt gcgagttgta aatgggattc caacacgaac 3000

3060

3120

3180

ggttctcaat gtttcccagc tggtatatgg ccctgaagga tcagatctgg ttttaatgaa 3240

gcttgccagg cctgctgtcc tggatgattt tgttagtacg attgatttac ctaattatgg 3300

3360

3420

ccagcatcat cgagggaagg tgactctgaa tgagtctgaa atatgtgctg gggctgaaaa 3480

gattggatca ggaccatgtg agggggatta tggtggccca cttgtttgtg agcaacataa 3540

aatgagaatg gttcttggtg tcattgttcc tggtcgtgga tgtgccattc caaatcgtcc 3600

3660

taaggtacca cagtcatag 3679

<210> 14

<211> 2730

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 14

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc tttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgga tccttatgtt tcagacaact tcgattcagt tctagaatgt ttgactcagc 780

aaattcacag gctcatcttt ctaacttgat ggtgaatatg gaaattcagc taaatggatg 840

ttaataaaat tcaaacgttt taaggacaga tggaaatgac agaattttaa ggtaaaatat 900

atgaaggaat ataagataaa ggatttttct accttcagca aaaacatacc cactaattag 960

taaaattaat aggcgaaaaa aagttgcatg ctcttatact gtaatgatta tcattttaaa 1020

actagctttt tgccttcgag ctatcggggt aaagacctac aggaaaacta ctgtcgaaat 1080

cctcgagggg aagaaggggg accctggtgt ttcacaagca atccagaggt acgctacgaa 1140

gtctgtgaca ttcctcagtg ttcagaagtt gaatgcatga cctgcaatgg ggagagttat 1200

cgaggtctca tggatcatac agaatcaggc aagatttgtc agcgctggga tcatcagaca 1260

ccacaccggc acaaattctt gcctgaaaga tatcccgaca agggctttga tgataattat 1320

tgccgcaatc ccgatggcca gccgaggcca tggtgctata ctcttgaccc tcacacccgc 1380

tgggagtact gtgcaattaa aacatgcgct gacaatacta tgaatgacac tgatgttcct 1440

ttggaaacaa ctgaatgcat ccaaggtcaa ggagaaggct acaggggcac tgtcaatacc 1500

atttggaatg gaattccatg tcagcgttgg gattctcagt atcctcacga gcatgacatg 1560

actcctgaaa atttcaagtg caaggaccta cgagaaaatt actgccgaaa tccagatggg 1620

tctgaatcac cctggtgttt taccactgat ccaaacatcc gagttggcta ctgctcccaa 1680

attccaaact gtgatatgtc acatggacaa gattgttatc gtgggaatgg caaaaattat 1740

atgggcaact tatcccaaac aagatctgga ctaacatgtt caatgtggga caagaacatg 1800

gaagacttac atcgtcatat cttctgggaa ccagatgcaa gtaagctgaa tgagaattac 1860

tgccgaaatc cagatgatga tgctcatgga ccctggtgct acacgggaaa tccactcatt 1920

ccttgggatt attgccctat ttctcgttgt gaaggtgata ccacacctac aatagtcaat 1980

ttagaccatc ccgtaatatc ttgtgccaaa acgaaacaat tgcgagttgt aaatgggatt 2040

ccaacacgaa caaacatagg atggatggtt agtttgagat acagaaataa acatatctgc 2100

ggaggatcat tgataaagga gagttgggtt cttactgcac gacagtgttt cccttctcga 2160

2220

aaatgcaaac aggttctcaa tgtttcccag ctggtatatg gccctgaagg atcagatctg 2280

gttttaatga agcttgccag gcctgctgtc ctggatgatt ttgttagtac gattgattta 2340

cctaattatg gatgcacaat tcctgaaaag accagttgca gtgtttatgg ctggggctac 2400

actggattga tcaactatga tggcctatta cgagtggcac atctctatat aatgggaaat 2460

gagaaatgca gccagcatca tcgagggaag gtgactctga atgagtctga aatatgtgct 2520

ggggctgaaa agattggatc aggaccatgt gagggggatt atggtggccc acttgtttgt 2580

gagcaacata aaatgagaat ggttcttggt gtcattgttc ctggtcgtgg atgtgccatt 2640

ccaaatcgtc ctggtatttt tgtccgagta gcatattatg caaaatggat acacaaaatt 2700

attttaacat ataaggtacc acagtcatag 2730

<210> 15

<211> 3250

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 15

cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 60

tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 120

ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 180

ctggcctttt gctcacatgc gcgttgacat tgattattga ctagttatta atagtaatca 240

attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 300

aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 360

gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 420

taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac 480

gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt 540

cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg 600

cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 660

attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 720

aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 780

agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac 840

ctccatagaa gacaccggga ccgatccagc ctccgcggcc gggaacggtg cattggaacg 900

cggattcccc gtgccaagag tgacgtaagt accgcctata gactctatag gcacacccct 960

ttggctctta tgcatgctat actgtttttg gcttggggcc tatacacccc cgcttcctta 1020

1080

tcccctattg gtgacgatac tttccattac taatccataa catggctcta gacttaaggc 1140

agcggcagaa gaagatgtag gcagctgagt tgttgtattc tgataagagt cagaggtaac 1200

tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc tgagcagtac tcgttgctgc 1260

1320

1380

ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 1440

gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 1500

aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 1560

1620

aattcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc 1680

gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc 1740

acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 1800

gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt 1860

cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg 1920

cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg 1980

agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 2040

aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag 2100

gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 2160

ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 2220

ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 2280

aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 2340

ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 2400

tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc 2460

cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc 2520

aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgcgt cagaccccgt 2580

agaaaagatc aaaggatctt cttgagatcc ttttttttctg cgcgtaatct gctgcttgca 2640

aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 2700

ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 2760

gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 2820

aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 2880

aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 2940

gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3000

3060

aacaggagg cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3120

cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3180

cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3240

tgctcacatg 3250

<210> 16

<211> 4108

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 16

cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 60

tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 120

ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 180

ctggcctttt gctcacatgc gcgttgacat tgattattga ctagttatta atagtaatca 240

attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 300

aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 360

gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 420

taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac 480

gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt 540

cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg 600

cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 660

attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 720

aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 780

agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac 840

ctccatagaa gacaccggga ccgatccagc ctccgcggcc gggaacggtg cattggaacg 900

cggattcccc gtgccaagag tgacgtaagt accgcctata gactctatag gcacacccct 960

ttggctctta tgcatgctat actgtttttg gcttggggcc tatacacccc cgcttcctta 1020

1080

tcccctattg gtgacgatac tttccattac taatccataa catggctcta gacttaaggc 1140

agcggcagaa gaagatgtag gcagctgagt tgttgtattc tgataagagt cagaggtaac 1200

tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc tgagcagtac tcgttgctgc 1260

cgcgcgcgcc accagacata atagctgaca gactaacaga ctgttccttt ccatgggtct 1320

tttctgcagt caccgtcctt gacacgaagc ttatcgatat gggaaaaatc agcagtcttc 1380

caacccaatt atttaagtgc tgcttttgtg atttcttgaa ggtgaagatg cacaccatgt 1440

cctcctcgca tctcttctac ctggcgctgt gcctgctcac cttcaccagc tctgccacgg 1500

ctggaccgga gacgctctgc ggggctgagc tggtggatgc tcttcagttc gtgtgtggag 1560

acaggggctt ttatttcaac aagcccacag ggtatggctc cagcagtcgg agggcgcctc 1620

agacaggcat cgtggatgag tgctgcttcc ggagctgtga tctaaggagg ctggagatgt 1680

attgcgcacc cctcaagcct gccaagtcag ctcgctctgt ccgtgcccag cgccacaccg 1740

acatgcccaa gacccagaag gtaagcccac ctgggtggga tccagccatc ctcaagtggt 1800

ctctctcttg tgcatgtggg tgggccaagc agaaatcctg ccccatagtc tcctggctta 1860

caagtcagaa aagctccttt gcaccaaagg gatggattac atccccatct ctttgctaaa 1920

caaacatggg ctttggtgtc agacaaaagt gaagtcctgg ctttctcaca caccagctta 1980

gagagaaaag acttttaggt gaatgtggca ggaaagcgtg cttgctgggg caaaggcaga 2040

ttcattcttt ctcttcccag tatcagcccc catctaccaa caagaacacg aagtctcaga 2100

2160

tacatttgaa gaacgcaagt agagggagtg caggaaacaa gaactacagg atgtaggtcg 2220

acctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt 2280

gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 2340

ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 2400

ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 2460

ggcatgctgg ggagtcgaaa ttcagaagaa ctcgtcaaga aggcgataga aggcgatgcg 2520

ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc 2580

aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc 2640

cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa 2700

gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct 2760

ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac 2820

aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa 2880

tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac 2940

tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag 3000

3060

cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt tcattcaggg caccggacag 3120

gtcggtcttg acaaaaagaa ccgggcgcc ctgcgctgac agccggaaca cggcggcatc 3180

agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc 3240

cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc 3300

ttgatcagat cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt 3360

tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct 3420

tgctgcgtca gaccccgtag aaaagatcaa aggatcttct

3540

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 3600

tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 3660

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 3720

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 3780

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 3840

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 3900

3960

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 4020

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 4080

ggccttttgc tggccttttg ctcacatg 4108

<210> 17

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic primer

<400> 17

agctggcaat tccggttcgc ttgctgcgtc agaccccgta 40

<210> 18

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence description: Synthetic primer

<400> 18

tacggggtct gacgcagcaa gcgaaccggga attgccagct 40

<210> 19

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic primer

<400> 19

ctaatccata acatggctct agacttaagg cagcggcaga 40

<210> 20

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic primer

<400> 20

tctgccgctg ccttaagtct agagccatgt tatggattag 40

<210> 21

<211> 933

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 21

atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60

aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120

accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180

gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240

tccagcagtc ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt 300

gatctaagga ggctggagat gtattgcgca cccctcaagc ctgccaagtc agctcgctct 360

gtccgtgccc agcgccacac cgacatgccc aagacccaga aggtaagccc acctgggtgg 420

gatccagcca tcctcaagtg gtctctctct tgtgcatgtg ggtgggccaa gcagaaatcc 480

tgccccatag tctcctggct tacaagtcag aaaagctcct ttgcaccaaa gggatggatt 540

acatccccat ctctttggtc actctgcatt gcaaatttcc cctcccaccg ctatggacga 600

tgtgatgatt ggaagatgtt acaaaacagt ggctaaacaa acatgggctt tggtgtcaga 660

caaaagtgaa gtcctggctt tctcacacac cagcttagag agaaaagact tttaggtgaa 720

tgtggcagga aagcgtgctt gctggggcaa aggcagattc attctttctc ttcccagtat 780

cagcccccat ctaccaacaa gaacacgaag tctcagagaa ggaaaggaag tacatttgaa 840

gaacgcaagt agctttttct cctttattta taggaagtac atttgaagaa cgcaagtaga 900

gggagtgcag gaaacaagaa ctacaggatg tag 933

<210> 22

<211> 858

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 22

atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60

aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120

accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180

gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240

tccagcagtc ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt 300

gatctaagga ggctggagat gtattgcgca cccctcaagc ctgccaagtc agctcgctct 360

gtccgtgccc agcgccacac cgacatgccc aagacccaga aggtaagccc acctgggtgg 420

gatccagcca tcctcaagtg gtctctctct tgtgcatgtg ggtgggccaa gcagaaatcc 480

tgccccatag tctcctggct tacaagtcag aaaagctcct ttgcaccaaa gggatggatt 540

acatccccat ctctttgcta aacaaacatg ggctttggtg tcagacaaaa gtgaagtcct 600

ggctttctca cacaccagct tagagagaaa agacttttag gtgaatgtgg caggaaagcg 660

tgcttgctgg ggcaaaggca gattcattct ttctcttccc agtatcagcc cccatctacc 720

aacaagaaca cgaagtctca gagaaggaaa ggaagtacat ttgaagaacg caagtagctt 780

tttctccttt atttatagga agtacatttg aagaacgcaa gtagagggag tgcaggaaac 840

aagaactaca ggatgtag 858

<210> 23

<211> 153

<212> Protein

<213> Homo sapiens

<400> 23

Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe

1 5 10 15

Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu

20 25 30

Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala

35 40 45

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe

50 55 60

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly

65 70 75 80

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys

85 90 95

Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu

100 105 110

Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp

115 120 125

Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly

130 135 140

Ser Ala Gly Asn Lys Asn Tyr Arg Met

145 150

<210> 24

<211> 462

<212> DNA

<213> Homo sapiens

<400> 24

atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60

aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120

accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180

gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240

tccagcagtc ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt 300

gatctaagga ggctggagat gtattgcgca cccctcaagc ctgccaagtc agctcgctct 360

gtccgtgccc agcgccacac cgacatgccc aagacccaga aggaagtaca tttgaagaac 420

gcaagtagag ggagtgcagg aaacaagaac tacaggatgt ag 462

<210> 25

<211> 158

<212> Protein

<213> Homo sapiens

<400> 25

Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe

1 5 10 15

Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu

20 25 30

Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala

35 40 45

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe

50 55 60

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly

65 70 75 80

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys

85 90 95

Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu

100 105 110

Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp

115 120 125

Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr

130 135 140

Lys Ser Gln Arg Arg Lys Gly Ser Thr Phe Glu Glu Arg Lys

145 150 155

<210> 26

<211> 477

<212> DNA

<213> Homo sapiens

<400> 26

atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60

aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120

accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180

gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240

tccagcagtc ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt 300

gatctaagga ggctggagat gtattgcgca cccctcaagc ctgccaagtc agctcgctct 360

gtccgtgccc agcgccacac cgacatgccc aagacccaga agtatcagcc cccatctacc 420

aacaagaaca cgaagtctca gagaaggaaa ggaagtacat ttgaagaacg caagtag 477

<210> 27

<211> 137

<212> Protein

<213> Homo sapiens

<400> 27

Met Ile Thr Pro Thr Val Lys Met His Thr Met Ser Ser Ser His Leu

1 5 10 15

Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala

20 25 30

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe

35 40 45

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly

50 55 60

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys

65 70 75 80

Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu

85 90 95

Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp

100 105 110

Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly

115 120 125

Ser Ala Gly Asn Lys Asn Tyr Arg Met

130 135

<210> 28

<211> 414

<212> DNA

<213> Homo sapiens

<400> 28

atgattacac ctacagtgaa gatgcacacc atgtcctcct cgcatctctt ctacctggcg 60

ctgtgcctgc tcaccttcac cagctctgcc acggctggac cggagacgct ctgcggggct 120

gagctggtgg atgctcttca gttcgtgtgt ggagacaggg gctttttttt caacaagccc 180

acagggtatg gctccagcag tcggagggcg cctcagacag gcatcgtgga tgagtgctgc 240

ttccggagct gtgatctaag gaggctggag atgtattgcg cacccctcaa gcctgccaag 300

tcagctcgct ctgtccgtgc ccagcgccac accgacatgc ccaagaccca gaaggaagta 360

catttgaaga acgcaagtag agggagtgca ggaaacaaga actacaggat gtag 414

<210> 29

<211> 195

<212> Protein

<213> Homo sapiens

<400> 29

Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe

1 5 10 15

Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu

20 25 30

Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala

35 40 45

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe

50 55 60

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly

65 70 75 80

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys

85 90 95

Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu

100 105 110

Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp

115 120 125

Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr

130 135 140

Lys Ser Gln Arg Arg Lys Gly Trp Pro Lys Thr His Pro Gly Gly Glu

145 150 155 160

Gln Lys Glu Gly Thr Glu Ala Ser Leu Gln Ile Arg Gly Lys Lys Lys

165 170 175

Glu Gln Arg Arg Glu Ile Gly Ser Arg Asn Ala Glu Cys Arg Gly Lys

180 185 190

Lys Gly Lys

195

<210> 30

<211> 588

<212> DNA

<213> Homo sapiens

<400> 30

atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60

aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120

accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180

gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240

tccagcagtc ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt 300

gatctaagga ggctggagat gtattgcgca cccctcaagc ctgccaagtc agctcgctct 360

gtccgtgccc agcgccacac cgacatgccc aagacccaga agtatcagcc cccatctacc 420

aacaagaaca cgaagtctca gagaaggaaa ggttggccaa agacacatcc aggaggggaa 480

cagaaggagg ggacagaagc aagtctgcag atcagaggaa agaagaaaga gcagaggagg 540

gagattggaa gtagaaatgc tgaatgcaga ggcaaaaaag gaaaatga 588

<210> 31

<211> 402

<212> DNA

<213> Homo sapiens

<400> 31

atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60

aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120

accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180

gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240

tccagcagtc ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt 300

gatctaagga ggctggagat gtattgcgca cccctcaagc ctgccaagtc agctcgctct 360

gtccgtgccc agcgccacac cgacatgccc aagacccaga ag 402

<210> 32

<211> 1505

<212> DNA

<213> Homo sapiens

<400> 32

gtaagcccac ctgggtggga tccagccatc ctcaagtggt ctctctcttg tgcatgtggg 60

tgggccaagc agaaatcctg ccccatagtc tcctggctta caagtcagaa aagctccttt 120

gcaccaaagg gatggattac atccccatct ctttggtcac tctgcattgc aaatttcccc 180

tcccaccgct atggacgatg tgatgattgg aagatgttac aaaacagtgg ctaaacaaac 240

atgggctttg gtgtcagaca aaagtgaagt cctggctttc tcacacacca gcttagagcc 300

cttggcaaat aatgtgatgt acccaagcct cagtttcatc agtaacattg ggataataat 360

aatatctacc acatcagttt gttgtcaaaa ttaagtagct catgcatata ctttgagatg 420

cttttcacat gcctgcataa agtaattgtt ggaccatcgt taatgtctgc cataattgca 480

cttaataaca aagcttgtaa cctttcaagt tctgagattc tacaatcttc caaagaaaat 540

aaaaggctaa tgggaactat tcaaaattca tattcagtag caagcataat taaacatgaa 600

660

aatctgcagg catcattttc taagagctag actgtaaaca aacctcagag gtaccaacta 720

tgccatcagt agtacataaa acatctgatg cacatttagt cacttgatcg atttctcttg 780

aatgagtgaa cgaatgaaca aatgaatata agagattaaa attttagcca ttaagtagaa 840

agaataagaa ctaaagagaa ggtaaaggag gaaaaagaga aggcaaggaa gttgagtaag 900

ggaagaaata gctctcgttt aagtattttg gggactctgt tgaaaaaaga aatgccaaca 960

1020

tcaagaagtt caccatgaca ccttcgctgc ttcctggtct taaacctcag ctgaggctgg 1080

aagaggacca tggtggctta ttggagatgt gaccccaggg agcccctctg aaggatggaa 1140

ggggactggg caagacccaa cacacacaga acacagtagc cactggccag gcaggaagca 1200

aggatctcag aaaagacttt taggtgaatg tggcaggaaa gcgtgcttgc tggggcaaag 1260

gcagattcat tctttctctt cccaggtgac ccagcgcctc ttggtttcta actggggagg 1320

gggtaggtgt caagagatga gtcccaaagt tctggaatgg tgggtcttgt gactgaggtc 1380

tagacccctc tccagcatga gtgctgtctc ctgcatcata tggagcctgg gcattctgag 1440

ctcattcaaa gggacaccat gggaaccact tgttctcaat gcaattattt ttgtgatgtt 1500

tacag 1505

<210> 33

<211> 75

<212> DNA

<213> Homo sapiens

<400> 33

tatcagcccc catctaccaa caagaacacg aagtctcaga gaaggaaagg aagtacattt 60

gaagaacgca agtag 75

<210> 34

<211> 15250

<212> DNA

<213> Homo sapiens

<400> 34

aggacaggag gattaaacag acagaggcaa ggatgatgag agaggagcag acagcaagaa 60

tgaaaagcag aaaatacaat agaggaaatg aagaaaagta ggcctgctgg agctagatga 120

tgatgtgatg gaaatagaag taacctttta gagaatctcg ctaagaaaca tggagaaaac 180

ggaaaagaaa aatgtaatgc cctagaaagc gcaaagaaag acagtggcaa aaatgaaaaa 240

aaaaaataaa aattataaaa gaggcaaaaa aagacacact attctctgcc tctaaaacac 300

360

tgaatctgtt atggaagaat tgcctatgtc aatatatgtt cagagttaaa tattagcccc 420

aaatgctcag caagactgaa ttgtgtcata gaagttccca gattcccttt tcccgcaatg 480

tcattggagg ctgcatttct tagtcaagtc cagggtttag gccaaagggc atccggtatt 540

gcctaaaacc ctgtgaggtc tgtgaggtaa cttttgagaa gaggtcactg cactcttcat 600

cttttttgca ctttggaatc agatataaaa gatgtataag tttgctaggg ctgccataac 660

aaagtatcat aggctaggta gtttaaacca cagaaattga ttttttcata gttctgggag 720

ttgaaagtcc aaaatcaaag tatcagccct tgcaagggcc ttagagaagg ctctgtcatg 780

ggctcctccc ctcggcttgt aggtggcctc cttcttctcc ccctgtgtct tcacttcatc 840

ttccctccat acatatctct gtgtctaaac atcctctgtg tgaaacaaca ccagccaggt 900

tggatttggg cccaccccac tgacctcatt ttaacttaat tatctctgta aagactctgt 960

ctccaaatac agtcatattt tgacgtactg ggagttaggg cttcaacaca tgaatttgga 1020

cacaattcag ccagtgacag aagacttctg atctctgatg ataaccactg cattttgatt 1080

acagctccta gaaaacactc ccctccacca ccccaccaca gatctatttt tatatctgaa 1140

accctgagtt tctgctccat gagaacccca ggaacatact atgttagatc tggaagaagc 1200

ctcagaaatc cccttatttt gaagactagg acactgagat ccagaagtgg gtaaagatgt 1260

gcttgggttc taagctgctc ttcttttggc caggagacaa cagcacataa tcaaagtggg 1320

tcaactaaga aagaattcca gaaggaaaag aggggcaga aatgaaggga gagaatgaga 1380

gcaaaagtgc tggatttccc tgagggtgaa gaaaagttaa atagaatcac agaattcaga 1440

ttttagagat cttctccttc agatcccttg gtttaatcag taggattggg gtcttcatag 1500

ataataaagc aaaaactctc gccatcctcc aagttgtgaa ttagaagagc tgagaaaggg 1560

tacaagacgg aagttctcta ccaaacaaat ggtgacattt tggggtaaga atatgactaa 1620

cccagaagtg aagcatttca tccaagtagt ctattttgaa gatgtcatgg tataaaggaa 1680

cctcctttct gcctggtcct ccatgcctct gccatgcttt ttactccagg atcacccttt 1740

ctagtggttc actgaaaacc caggattact taaatatgat ggacatgttc acggctcaat 1800

ccaggaggaa aaggtcgaac tgaaagcatg ccaaagcccc acatgggagc caagccactg 1860

ctgctgtggt tgcaaagtgg atcctggctt atcagagcag agagaagcca ggctcgtgcc 1920

ttagcccaag tggccagtca ccttattcag gagatactaa gttctccagc taagacatcc 1980

atgctttggg accagctgca gacagaagcc aattcctact acaaccatca ccttagagta 2040

gcatatagac acagatggct cttcaaagga ccacagttcc atggaataac taagaattca 2100

tgtcctgtgg aaaggtttga ataaactata attataccca atcataaatt tcattcaaga 2160

aaggagggag ggagggaggg 2220

aaggaaggaa ggaaggaagg aaggaaggaa ggaaggaagg aaggaaggaa ggaaagggaa 2280

ggaagaacaa aaagactttc tagttaaaga atgcttaact agcaaactat gtactataag 2340

acagttcttt tcggaatgag ttttatcaac tctaaagcaa ttatcttgaa tgcctacatg 2400

tgattactga ataatatgaa ccaagaaaac agaaagaatc tatattatct ttccatttcc 2460

ttctttccag tatcaatacc caagcctcta gtgatacatg gcatataatg ttggatggat 2520

ggatggatgg atggatggat ggatggatgg atggatggat gaatggatgg ttggatggac 2580

aaatgagtaa cataggctga tgaatagtgg tagaaagaca caccataaaa acaagtggca 2640

cttctgagat gaaatgattc ctattctcct acacaagaca gtgaggcaag tacagagtaa 2700

2760

tggccaaatt gaaattgttc aaggacctat tgctacaggt ggcaactggc taagaatttc 2820

atagtgaata ttaacacct attactcccc ttaatgtttc tttgaagtaa gcagaatatt 2880

aataatcatt taaaattcca gtgtttcaac ttcaattgtt tcctagggca aattgataat 2940

tgtgtgtaaa actaattgga atatgtatgg aataatcatc ctgaaataaa attggtgaaa 3000

agtatttgtt attgggcatc tacaatgtgc aaacctctgt actaggcatg aacaagagtt 3060

ataagcattg gagaggctaa aatatagtcc ttaaggctgg gcacagtggc tcatgcctgt 3120

aatcctagca ctttgggagg ccaaggcggg cagattgcct gagctcagga gttcaagacc 3180

agcctgggca acatagcgaa accccatctc tactaaaaat acaaaaaaat tacctgggca 3240

tggtggcacg cacctgtaat cccagctact caggaggctg aggcatgaga attccttgaa 3300

cctgggaggc agaggttgca gcgagccgag atcctgccgc tgcatcccag cttgggtgac 3360

agagtgagac tctgtctcaa aaaaaaatta aataataaat aaatagtaaa atacagtcat 3420

taagagtaca aaatgtagat tcagactacc tgggttcaaa tcttggctct tacttgcatt 3480

gtggctttgg gcagatcatg taacttatgt gtgcctcagt ttcctcatct gttaaatagg 3540

3600

3660

tcagagacac atattacagc acttaaaatg gtatctagca cttagtaagc attattcaag 3720

aaaattggag ctatgtgaaa aagaagttgg 3780

tctcctgaag tagaagccag tcttgtgtca ccaaaaactt caagcccaag cttgccaacg 3840

cttttccatg atgtggtagt agagtttcaa gcatgtggta ggataagaga actcaatgac 3900

ctaagaacca ttccaaccca gagaacccct ggttctatga ataattccaa cttaaatagg 3960

tagcttggct ctcccaagtg agagccattg cttctgtttc cgggtcatat aatgaacttt 4020

4080

tggtgcctgg ggaaaattta attgtagtat gaactccagt tattggtagt cttaagtaaa 4140

attgccaaaa taaatagaaa tgcaggatat ttctgggctc acacagcttc cgggacactt 4200

tagtttcttg ggctgccaat ccagtgcctt tcacaagcat ttgatctttt ttcaaacatc 4260

tcttgaaaac aaacaaaacc tcacacagct tctaatgtgt gcactgttcg aatgtaaggg 4320

tggaaaagga ggcaaagaaa tgagctccca aagagcaatt ccccttctct cgcctccatc 4380

ccttgacgac ctccctccca ctaaagggaa acattgtttt cttaggtaat aaattctgca 4440

atttctcaag tccattaaca tccactgggc aagatgagat ctattctttt tattgccca 4500

taggaaaaga atagtgcttt tttgcaatat tcactagata acacagagtt gacttttaat 4560

ccaagggcaa cattgatagt ctctagttaa aggggaagcc ttcaggagca atgaaaagat 4620

taatagtttt agatgaagca gaatccaaat ccctttttat gagttttgaa atatccagtt 4680

tgtatgctca cctcaatact taaagcccag ttactgattc ctttggccta agcaagacag 4740

gtcaattttt aaagagggag tagctgaggt tagcaaaaat tctccaggtc cacaaaactt 4800

ccagacctgc aaggtgaaaa tcagcttttc tgtcatccct aaaggcctaa ctggaatcag 4860

aacttttccc tgatgcccac atatttggag gtcctttttt aatgggactc cttaatgcct 4920

ttagtgccat cccattttca tccagtgtcc aaaagaaatg atttaaaaat ataaacgtat 4980

5040

gggaaaagag gtttatgagt ccatgtctga ttcttccaga gagcccctaa gaaagttctt 5100

atcataccag gaactcaatt ataactttca ttgcctattg ttagatgagt aacaggagct 5160

agaaaacatt ttggaaattc ccatctttat ttttttaact aatatgatta tagttttaag 5220

aaccattggt caagaagcta actttttaaa aagtggaagt atgatggtta gaaataagaa 5280

tgctaaaggt gcatcaagct gattttaatt ctaaatgtcc ttggcagcaa tttagaatct 5340

gtaataaact acaccaaaca gttttgaggg gaaggggatt agtttctccc cttccttcgt 5400

gtgtgtgtgt gcgcgtgtgt gtgtgtgcac ctttgtgttc tagcattgtt gcacccatta 5460

cagagctggg gggaactatt ttccaaaatt ataggtgaga acagtttctt ggattgtctt 5520

tcagtgaagg taaattcctc tgtaaaaact aaccatcatt cagtaaaaac tgcaggattc 5580

ctttgtcttc tcaaaagcct gtttctcatc ctaaattaaa aattattcag gaaatagaga 5640

ggacattatt ggaggggtgg aaataagttg gttttctttt tattgtatct tttgaggatc 5700

cagggacttc taccattttcc catctaacat acagagaagg attctctagg tccctgtcta 5760

tagactgcag taactttcct atagaaccaa tttgcaattt tagaaatttc taggtctaat 5820

tattgaccca ttacaaccaa aggtcaatgc atccagccaa tcttccttct atcatcccct 5880

gcccttactt ctattaggga ctgggattac aggcaaaacc catcaaatgc ctcttctacc 5940

actttcccat ttcttaacca ttagcctcta acttcctcta ttcagtttct catatgcttt 6000

catgcccatt gggtcagata aaggaacatt catttatttg agtaggcatc tgttatgatc 6060

actccggaaa aaagatgaca atgggttacc ttgtcctcct gggcttctct aactgacatg 6120

gtcaaaatgc ccatatgaag ataagatgtt aagagcaaga tttatgaaaa gctgagtatg 6180

atggcagctc ttgtctcata aaataactcg aaagttccca gtgaaagacc aagaaatttt 6240

acatcaaacc caaaccggcc aaatggtcca agcttccaag ctgggatcca tggctaaagt 6300

ttctacaaaa ttctgggtac aatgtataaa cattcacttg gggctttctg tctagccagc 6360

accaagaggt caagtaatca aggaccaact agccctgcca tctgtgaaaa tatgtgctat 6420

6480

tcaatcagcc gacaattgta gagttggggga 6540

tgtgcaatgt ttatgtcatg gtgtaagtat gtggcatgct tgactagctt gtgaggcact 6600

ggaagactag aaggaatgaa aaatatgaat gaatcaataa atgcatagta taattactgt 6660

tattttgtca gtattgtttt acctaggtca ctattgaatg ctctgatttg tctcttttata 6720

aataataata tgttttcttc ttcaaaagaa cactaggatg aaggtagagg tgcttttggc 6780

acaatgccac aattctgatt tttttaaaac tgtatgcatg cataaaatgt tcttgagcca 6840

ttctctgcct tggaatagca ctggctggca ttctgcatgt ttacttttat atgctgaagg 6900

cccccatcaa cctcaaacag aggcaaatca atttaacttc tcatagtgtt attttgttca 6960

tcctaaaagt tcaagagagc cttccaaact tccaaaattt ctctcaattc agtgaggagg 7020

aaaattcaga acacagcatt tgaatgttct gcccagattt gtcacacaca caaggaatga 7080

gtgaaagagg gcaacaccct ttcctcctaa ccctgtgaac tcatcactat tgcattgaaa 7140

tgacaccaaa aggtaaaaac cctaggcctc acatctccca agaacactgc aataggagtt 7200

actgcataca ccagtttaag taactctagc ataaattgta tgtcagatga aacaatggca 7260

ttttgggaggc ttaagagaaa aagaataatc aaatccagtt tttaggtact aatgtgctga 7320

atctttagca catagcagca aaattgctag aatctggtgt ttcacttttt aaaataccac 7380

atttgaacct ttcagcaatt ccaaaatcaa ctccctctgc gaaagataat aagcttaaac 7440

attttttaaa tttaaaaatg taacacaaac aaacagctaa gcaaacaagc tgcccataaa 7500

atcaacagtc tggggagccc tgatcctgaa gtattttaca acatccttca tgactattaa 7560

aggcaacata aacacctctt gtcagcaagg gaaactaccc ttggcatttt tttttctttg 7620

ttccccaggc tttttaaacca ttttgataga gattttttac atcacaggca gaaatatttg 7680

aaatagagtc aggtggtagt ctttaaaaga gtaagaaagt tgctaagtca agataatctt 7740

ggaataaagt cctctgattc ctggggattc ctagggatgc cccagtcact agaaaacaga 7800

gctgtaagtc cactctccca gcactcaacg gagctccgga aaccaaggag ctagctactg 7860

tttccccaca ttcagccaga gaaagggcag cactctagca tgcaaactgc tttgacaata 7920

gtaacaatta aaaagtaaat taaaaagaat cataatagct gatattgatt aggtacttgc 7980

cctgtggcaa gagctatagg gaatcacctc atttaatctt cacatgaagc ttgcagagtg 8040

8100

cttgccaacg tcttgggcta acaagcggtc aagcagaatc caaacccgag atagatagac 8160

cacagtgtgc taatcaagca ctgcactctc tcctgcattt cttagttgat atttaccata 8220

tacaatctgt cacttgtatg agatggcagg gggttctgtg ctatttgtcc ttgtagagaa 8280

taccacagga agaaagtaag cagccatgca atatttgctg ttgacctgaa ctccattcca 8340

tcattcctgc aggaaattcg catccattaa atgagcattt cctggtttgc cactttgctc 8400

aaacactttg cttggatctg gagaggatat agaagtgaag gaaatatgct acctgctctc 8460

aaggaactta tgttttagtg gagagacaaa catgcagaat ttactctaca gaacatcaat 8520

gcttgagcaa atgtagaccc agagaggggct cttacagcac acaagccaga acagactgat 8580

ggtgctaaca attaggttca aggtttttct aaacagtaga ctctcctgca tacaactata 8640

ccgcatgcca ggtaaatgac tgaggggttat tacatccaat tataacacca ctgtgatgta 8700

ggtgctctta ccccacactt tcattttaca gaagaggaaa ttgaggacag cacaatgtag 8760

tgattatcaa aggtcacacg actactgtgt gggagagcta ggatttaaac cagatgcata 8820

agatgaggtc ctccaagaaa cagaagatga gaaggtgtta aatgagcagg ggttttatta 8880

gggggaatta atgtgtgaac agaaataggg gaggataggc aaagccatca gattgcaagg 8940

caagcctaac cccaagggaa ggagagagag agagtagatt ggttggaaac atttttggtg 9000

ggtctatggt ctaaggaaag ttcagcaaag tcatcatgga gtttttgagc caaagttggg 9060

caatacagtt gcccaacaaa tttctgtgtt tctcagaaat aggtctgcct caatgtcccc 9120

accatacttg gtcactggct cttgggaggg gcctgccctg ttccaatcca ctagagccaa 9180

agaagagccg ttgtactggc aggggtggg ggaattccta caaccacata aaaagtgggg 9240

tgaggtttcc agaaaaaaac gtgatgctgg gctaaccaaa actgtgtcca gtaagtacat 9300

9360 atccctcact ctgttaaaga agcagccaca taaacaagga

gcaccttgtt ctttggtttt gaagtcacat cccaaagtgc tgagtagatc gcatgaccct 9420

cgctttgcct ggctgccaga gaggaaaggc tgatccaact ctcctggaat ttgaacttgt 9480

gattccctga agtaaagaga tatcaaagtt gatactgaga catctaaatc atcctccacc 9540

atttcacatg tccccaggcc aagccagcaa aattgctata gcacatccct ttcaacaggt 9600

aaagggctga tatctgagcc ctctttccaa tcatccactg ctcttttctt ctcattttgc 9660

cctttttggg agcaggtcaa tgctgagtta gtactttatg ctgtacaata agctgctgat 9720

attccatgct ggacagaatt ttcccagtat tttttataga gtgccaggct tttcctagac 9780

ttcatgtcat acaatactta acttgtttgg agtgggtgga gatggaaaca tagtctattg 9840

aaaacatcac tgcttcctcc ctgaagttta aagagcctat ttttatcctt ttagattcta 9900

tctctcaggc aaaatctcat aaagataagt ggggaggaaa aaaagggggt tataatacct 9960

agggagtttg cttttgctaa ttgaatactg tgctcctaga cttctataaa taccattaca 10020

aatgggtccc agcttgtggt aatactcacc ctcctcattg agtcttctgt cccatggcac 10080

agcctttccc tccaaactag catctacccc catctggaag catgggcagc tcatgatatt 10140

atcaactatt gctattggaa agtgatttgg acttgaaagc actagatatt ttttacctct 10200

tggggaggca gtttagcaga gtggttaact ggtgagctcc agaatcagaa ggaataggtc 10260

caaattccaa ccactattac atctccatca taagaaatta ggcaagttgt ttatcctaag 10320

tttcagattc cttaaagata aaacagtcaa gacagtagta cttatccctg agagaagtat 10380

aggaaacaag aaaatatatg caatttacat acatactaca atccccagca catgacaaat 10440

10500

acctcaagca cattactaaa tgttagcgag cttcagcttg tacgtgggac tgacaggaat 10560

10620

10680

atcttttttt tttttttttt tttttttttt tttttttttt ttggagacag agttttactc 10740

tgtcgccaag gctggaatgc agtggcacaa tctcagctca ctgcaacctc cgcctcctgg 10800

gttcaagcaa ttttcctgcc tcagcctcct gagtagctga aactacaggc acacgccacc 10860

acaccaggct aattttttgt atttttagta gagacggggt ttcaccatgt tggccaggct 10920

ggtctccagc tcctgacctc aagtgatctg cccacctcgg cctcccaaag tgctgggatt 10980

acaggtgtga gccaccacac ctggcacagt aatcttaatt gaaaagtctg tggatagctt 11040

tccaaaggaa agcttggagc ttggataaga accaagagat aatgggagaa ggtgaatggc 11100

ctcttcaggg ccttttctag caccctaaat atgcgtgtct gtccataatg ggtaatcata 11160

tatatcacaa atcaaaccct ccacaaactt atttcctaat gtgtttgtta acctttcctt 11220

ctaaagggta aacttcttta accaacccca gtgagctgga ggatcaatgt tttcttaata 11280

11340

11400

11460

tgcaccaaag gtgggtgcca gctctgatga cggctgcttc taacatgcct ccacttgccg 11520

cccattgtca agggtggctg gcgtaattaa gttaagacaa tgagcaaagc aacagatgca 11580

actgagacct agtccctgag tgcttttgtt ttgtcactgt cattgtctgc aacaaagaag 11640

tcacatgtga cagcctggga agagagccaa atgcaaacca

gaatggcctc caccgtgcac gtgtgtgcat gggaatcatg ctacttggta cagcatctgc 11760

11820

acagaccagg gaatccctcc gctcagactt tacactttat accttgtgct ttgagagaaa 11880

agaaaaagaa tctctctatt ggagacaaaa aataggatgt atgtggttgg tcaatctaac 11940

ctcaattctt tttgctatag ccccccgcta atttaaagag tgaagcatag atggtatctt 12000

aatgttttct tgtagaaatt tgggattaat ttggcttgag aggaagaatg gagattaaac 12060

gctttatgag gctttctttt aatttgttcc catttcattc ctgaatattt tcttagtttg 12120

ggcattgcag atgtttaaag aacttcttat tttgagctgg tatgcctctt aaacagaaaa 12180

12240

taggcagaga gatggcatcc ttaataatat ctattttgcg ggtttgatca gctacagacc 12300

12360

atgctattct gtggccccat gtgtccttct ctcatttttc tagagaactc ctataagaaa 12420

gcagaacacg gccaggcatg atggctcatg cctgtaatcc cagcacttca ggaggctgag 12480

gcaggcagat cacctgaggt caggagttca agaccagcct ggccaacatg gtgaaaccct 12540

atctctatta aaaatacaaa aaattagctg ggcatgatgg cgcgtgcctg taatcccagc 12600

tacttgggag gctgaggcag gagaatcact tgaactggga ggcaaaggtt gcagtgagcc 12660

12720

12780

gcctgtaata ccagcacttt gggaggccaa ggcgggtgga tcacaaggtc agaagttcga 12840

gaccaacctg gccaatatgg tgaaacccca tctctactaa aaagtacaaa aattagccat 12900

gcatggtggc aggcgtctgt aatcccagct actacagagg ctgaggcagg agaatcactt 12960

gaacccggga ggcagaggtt gcagtgagct gagaacgcgc cactgcactc cagcctgggt 13020

13080

actgggaact tagcagtaga tgctttgcac cacaacaaat gtatcttaag tggtcttttg 13140

13200

13260

agtgaatgtt tttacacaat gatgccatct tccttctact cagtcagtac aagatgttga 13320

13380

13440

taggtattat tcccaagggg tggcagttta tctttttccc cagccttcat attaaaacct 13500

ctcaccttct ccaggtctca ggtctgtgta atctcaaatg tgctttagct cctcacaata 13560

ttgtaactgt gtgggtgttc attaccttag ccagaagaca gtttacagat tccaggtctc 13620

13680

atccttctgt aggggctct ctggctagag ataaaaccaa aaaaagaagt acctcaggtt 13740

13800

13860

acttagttct gcacatcaca aggggtccc tcatactcat tcattcattt cactcatttt 13920

acagatattt attgagcacc tgcaataacc tgcacactgc tctagacact gggactataa 13980

cagtaaacag acagatacat ctctggtctc acagggcttc tattctaagc aaaactcaat 14040

atccaggccg ggtgcagtgg ctcatgcctg gaatgccagc actttgggag accaaggcca 14100

ggcagatcac ctgagcccac tagttgaaga ccagcctggg caatatagca aaaccccgtc 14160

tctacaaaaa aaaaaaaaaa aaaaaaaaaa aaattgtcaa ggcatggtgg catgcgcctg 14220

tggtcccagc tacttaggag gctgaggcag gaggattgtg taagcctggg aggcagaggt 14280

tgcagtgacc tgagatggca ccaccacact ccagcctggg caacagagtg agaccctgtc 14340

14400

ttaacaccaa attaggaacc caggactttt agtcttggca tggttacttt ccaataaaga 14460

tgacaatact aagaagagaa aaatgattta ataatgataa tagtggctaa tacttatgta 14520

14580

tgatcctata aggtagatat tattgttacc ctagtttata gatgaagaaa cggaaacaca 14640

agagattgcc actcatacaa gtttacacag ccagaaaata gaaaagctac gagttgagct 14700

cagcccagta tgtctatgat tttacagact caaaattaat tataagattt cctaatcttc 14760

gatttctgaa actctgcctt gctctagagg aaaacaagaa aaacaatgaa aaataaatgt 14820

ctctttttta caaaaattaa aacagaacaa actgcaataa aacaacagag gatgaatcca 14880

gaatgtgatt gatttttttt cttactagga aggatctag aggccagaag gctggatttt 14940

tcaggatctc ctttcaatca atgaatctgt gatagaagca gatgaatcaa atctcatctt 15000

tgtgtgatta taaagctgtc tgtggtattc acgccaccag gggtacatag aagatgcctg 15060

agtgaggttt ggcaaaagta ctaagggcct gtccacctat acatgccctt ctcaggaaaa 15120

ccaaggttca agctctctat tagctcaact ggtaaggcgt aagacatgga aggttgaggc 15180

ccaatgttag aaatagatgg atacataaaa cttcatcaag ttaatgtcac tttttctcct 15240

ttattatag 15250

<210> 35

<211> 60

<212> DNA

<213> Homo sapiens

<400> 35

gaagtacatt tgaagaacgc aagtagaggg agtgcaggaa acaagaacta caggatgtag 60

<210> 36

<211> 270

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 36

atgaacgcca aggtcgtggt cgtgctggtc ctcgtgctga ccgcgctctg cctcagcgac 60

gggaagcccg tcagcctgag ctacagatgc ccatgccgat tcttcgaaag ccatgttgcc 120

agagccaacg tcaagcatct caaaattctc aacactccaa actgtgccct tcagattgta 180

gcccggctga agaacaacaa cagacaagtg tgcattgacc cgaagctaaa gtggattcag 240

gagtacctgg agaaagcttt aaacaagtaa 270

<---

Claims (20)

1. Lyophilized pharmaceutical composition for the treatment of neuropathy, containing an effective amount of plasmid DNA, where this lyophilized pharmaceutical composition is obtained by lyophilization of a liquid composition, which before lyophilization contains: a) DNA pCK-HGF-X7, where pCK contains the nucleotide sequence of SEQ ID NO: 5 and where HGF-X7 contains the nucleotide sequence of SEQ ID NO: 13; b) potassium phosphate buffer with a pH in the range from 7.0 to 9.0; c) mannitol at a concentration in the range of 1 to 3% (w/v); d) sucrose at a concentration greater than 0.5% (w/v) and less than 1.1% (w/v) and e) NaCl in a concentration ranging from 0.1 to 0.9% (w/v). 2. The lyophilized pharmaceutical composition of claim 1, wherein at least 90% of the plasmid DNA in the liquid composition is supercoiled. 3. The lyophilized pharmaceutical composition according to claim 1, wherein at least 90% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved. 4. The lyophilized pharmaceutical composition according to claim 1, wherein at least 90% of the plasmid DNA remains supercoiled after storage at 25°C for 3-7 days after the lyophilized pharmaceutical composition is dissolved. 5. The lyophilized pharmaceutical composition according to claim 1, wherein at least 80% of the plasmid DNA remains supercoiled 30 minutes after the lyophilized pharmaceutical composition is dissolved, where the lyophilized pharmaceutical composition is stored at 40°C for 10 weeks before dissolution. 6. The lyophilized pharmaceutical composition of claim 1, wherein the lyophilized pharmaceutical composition contains supercoiled DNA in an amount of at least 90% of the total amount of supercoiled DNA in the liquid composition. 7. Lyophilized pharmaceutical composition according to claim 1, where this liquid composition contains potassium phosphate at a concentration in the range from 5 to 15 mm; this liquid composition contains a potassium phosphate buffer with a pH in the range from 7.0 to 8.0; while this liquid composition contains mannitol in a concentration in the range from 2 to 3% (wt./vol.); while this liquid composition contains sucrose in a concentration in the range from 0.75 to 1.1% (w/v); or while this liquid composition contains NaCl in a concentration in the range from 0.1 to 0.75% (w/v). 8. Dissolved composition for the treatment of neuropathy, obtained by dissolving a lyophilized pharmaceutical composition according to any one of paragraphs. 1-7 in water. 9. The dissolved composition of claim 8, wherein the light absorption of the dissolved composition at 450 nm is less than 0.002. 10. The dissolved composition according to claim 9, wherein the light absorption is measured on the day of dissolution. 11. The dissolved composition according to claim 9, wherein the light absorption is measured after storage of the lyophilized pharmaceutical composition for 10 weeks.

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