COMPOSITIONS AND METHODS FOR REGULATING PRODUCTION OF A FUSION PROTEIN AND RIBONUCLEIC ACID

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted via Patent Center to the United States Patent and Trademark Office as an XML Document file entitled “A8147625US-ST26.xml” created on 2022 Dec. 4 and having a size of 48,355 bytes. The information contained in the Sequence Listing is incorporated by reference herein.

TECHNICAL FIELD

The present disclosure generally relates to compositions and methods for regulating production of a fusion protein and ribonucleic acid (RNA). In particular, the present disclosure relates to compositions and methods for regulating gene expression and, therefore, production of a fusion protein and micro-RNA both of which relate to suppressing immune responses.

BACKGROUND

Bioactive molecules, including enzymes, receptors, receptor agonists and antagonists, are necessary for the homeostatic control of biological systems.

When bioactive molecules are over-expressed, under-expressed or mis-expressed, homeostasis is lost, and disease is often the result.

Known approaches to the treatment of conditions whereby bioactive molecules are over or mis-expressed are the commercially available pharmaceutical products that bind to and block the production or effectiveness of one or more bioactive molecules.

SUMMARY

Some embodiments of the present disclosure relate to one or more compositions that upregulate the production of a belatacept-similar protein (BSP). The BSP has a similar, substantially the same or the same biological function as belatacept when administered to a subject. In some embodiments the compositions comprise a vector of plasmid deoxyribonucleic acid (DNA) that includes an insert sequence of nucleic acids that encode for the production of the BSP and a backbone sequence of nucleic acids that facilitate introduction of the insert sequence into one or more of a subject's cells where the insert sequence is expressed and/or replicated. Expression of the insert sequence by one or more cells of the subject results in an increased production of the BSP by the one or more cells. In some embodiments of the present disclosure, the methods that upregulate the production of BSP relate to methods of manufacturing and administering the composition.

Some embodiments of the present disclosure relate to one or more compositions that upregulate the production of one or more sequences of micro-ribonucleic acid (miRNA). The sequences of miRNA may be complimentary to a sequence of target messenger RNA (mRNA) that encodes for translation of a target biomolecule and the miRNA can cause the target mRNA to be degraded or inactivated, thereby causing a decrease in production of the target biomolecule. In some embodiments of the present disclosure, the target biomolecule is a pro-inflammatory cytokine, such as tumor necrosis factor alpha (TNF-alpha). In some embodiments of the present disclosure, the target biomolecule is an anti-inflammatory cytokine. In some embodiments of the present disclosure the compositions comprise vector DNA that includes one or more an insert sequences of nucleic acids that encode for the production of miRNA and a backbone sequence of nucleic acids that facilitates introduction of the one or more insert sequences into one or more of a subject's cells where it is expressed and/or replicated. Expression of the one or more insert sequences by one or more cells of the subject results in an increased production of the miRNA and, therefore, decreased translation of the target cytokine by one or more of the subject's cells. In some embodiments of the present disclosure, the methods that upregulate the production of the one or more miRNA sequences relate to methods of manufacturing and administering the composition.

Some embodiments of the present disclosure relate to one or more compositions that upregulate the production of both the BSP and miRNA that degrades, or causes degradation of, or inactivates or causes the inactivation of the mRNA of the target biomolecule, such as a cytokine.

Some embodiments of the present disclosure relate to compositions that can be used as a therapy or a treatment for a subject that has a condition whereby the target biomolecule is over or mis-expressed.

Some embodiments of the present disclosure relate to a recombinant plasmid (RP). In some embodiments of the present disclosure, the RP comprises a nucleotide sequence of SEQ ID No. 1. The RP comprises a nucleotide sequence encoding one or more nucleotide sequences encoding for production of the BSP and production of a miRNA sequence that targets mRNA of the target biomolecule; and, an inverted terminal repeat.

Some embodiments of the present disclosure relate to a cassette, which may also be referred to as an insert, for use with an RP, wherein the cassette comprises a nucleotide sequence of SEQ ID No. 1. When the cassette with SEQ ID No. 1 is included in the RP and the RP is administered to a subject, one or more of the subject's cells will upregulate production of the BSP and the miRNA that degrades, or causes degradation of, or inactivates or causes the inactivation of the target mRNA of the target biomolecule. For example, the target biomolecule may be TNF-alpha.

Some embodiments of the present disclosure relate to a method of making a composition/target cell complex. The method comprising a step of administering a RP comprising SEQ ID No. 1 to a target cell for forming the composition/target cell complex, wherein the composition/target cell complex causes the target cell to increase production of BSP and one or more sequences of miRNA that decreases production of a target biomolecule. For example, the target biomolecule may be TNF-alpha.

Some embodiments of the present disclosure relate to a method of making an agent/target cell complex, the method comprising a step of administering a sufficient amount of an agent to a target cell whereby the agent/target cell complex is formed, wherein the agent/target cell complex increases the subject's production of the BSP and the agent/target cell complex also increases the subject's production of one or more sequences of miRNA that decreases the production of a target cytokine.

Some embodiments of the present disclosure relate to a pharmaceutical agent that comprises an agent, a pharmaceutically acceptable carrier and/or an excipient. Administering the pharmaceutical agent to a subject may increase the subject's production of the BSP, one or more sequences of miRNA that decreases the production of a target cytokine or both.

Some embodiments of the present disclosure relate to a method of treating a condition. The method comprises a step of administering to a subject a therapeutically effective amount of an agent that upregulates the subject's production of the BSP, one or more sequences of miRNA that decreases the production of a target cytokine or both.

Some embodiments of the present disclosure relate to a use of an agent for treating a condition, wherein the agent upregulates the subject's production of the BSP, one or more sequences of miRNA that decreases the production of a target cytokine or both.

Embodiments of the present disclosure relate to at least one approach for inducing endogenous production of the BSP and one or more sequences of miRNA that target and silence mRNA of TNF-alpha protein. A first approach utilizes gene vectors containing nucleotide sequences for increasing the endogenous production of BSP and one or more sequences of miRNA that target and silence the mRNA of TNF-alpha, which can be administered to a subject to increase the subject's production of the BSP and one or more sequences of miRNA. Without being bound by any particular theory, embodiments of the present disclosure may be useful for treating conditions wherein the subject's immune system has become dysregulated.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the present disclosure will become more apparent in the following detailed description in which reference is made to the appended drawings.

FIG. 1 is a schematic of a vector map that represents a plasmid vector, according to embodiments of the present disclosure.

FIG. 2 is a schematic that represents a first portion of a plasmid vector, according to embodiments of the present disclosure.

FIG. 3 is a schematic that represents a second portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the first portion of FIG. 2.

FIG. 4 is a schematic that represents a third portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the second portion of FIG. 3.

FIG. 5 is a schematic that represents a fourth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the third portion of FIG. 4.

FIG. 6 is a schematic that represents a fifth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the fourth portion of FIG. 5.

FIG. 7 is a schematic that represents a sixth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the fifth portion of FIG. 6.

FIG. 8 is a schematic that represents a seventh portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the sixth portion of FIG. 7.

FIG. 9 is a schematic that represents an eighth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the seventh portion of FIG. 8.

FIG. 10 is a schematic that represents a ninth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the eighth portion of FIG. 9.

FIG. 11 is a schematic that represents a tenth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the ninth portion of FIG. 10.

FIG. 12 is a schematic that represents an eleventh portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the tenth portion of FIG. 11.

FIG. 13 is a schematic that represents a twelfth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the eleventh portion of FIG. 12.

FIG. 14 is a schematic that represents a thirteenth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the twelfth portion of FIG. 13.

FIG. 15 is a schematic that represents a fourteenth portion of a plasmid vector, according to embodiments of the present disclosure, which is contiguous with the thirteenth portion of FIG. 14.

FIG. 16 is a schematic of an adeno-associated viral genome, according to embodiments of the present disclosure.

FIG. 17 is an image of belatacept-similar protein analysis obtained from muscle samples of mice treated with an embodiment of the present disclosure.

FIG. 18 is a scatter plot of human IgG data over time taken from muscle samples of mice treated with an embodiment of the present disclosure.

FIG. 19 is an image of tumor necrosis factor alpha protein analysis obtained from muscle samples of mice treated with an embodiment of the present disclosure.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the meanings that would be commonly understood by one of skill in the art in the context of the present description. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, reference to “an agent” includes one or more agents and reference to “a subject” or “the subject” includes one or more subjects.

As used herein, the terms “about” or “approximately” refer to within about 25%, preferably within about 20%, preferably within about 15%, preferably within about 10%, preferably within about 5% of a given value or range. It is understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

As used herein, the term “activity” is used interchangeably with the term “functionality” and both terms refer to the physiologic action of biomolecule.

As used herein, the term “agent” refers to a substance that, when administered to a subject, causes one or more chemical reactions and/or one or more physical reactions and/or or one or more physiological reactions and/or one or more immunological reactions in the subject. In some embodiments of the present disclosure, the agent is a plasmid vector. Agent may be used interchangeably with the terms “compound” and/or “composition”.

As used herein, the term “ameliorate” refers to improve and/or to make better and/or to make more satisfactory.

As used herein, the term “biomolecule” refers to a carbohydrate, a protein, an amino acid sequence, a nucleic acid, a lipid, a primary metabolite, a secondary metabolite or another metabolite that is found within a subject. A biomolecule may be endogenous or exogenous to a subject.

As used herein, the term “cell” refers to a single cell as well as a plurality of cells or a population of the same cell type or different cell types. Administering an agent to a cell includes in vivo, in vitro and ex vivo administrations and/or combinations thereof.

As used herein, the term “complex” refers to an association, either direct or indirect, between one or more particles of an agent and one or more target cells. This association results in a change in the metabolism of the target cell. As used herein, the phrase “change in metabolism” refers to an increase or a decrease in the one or more target cells' production of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), one or more proteins, and/or any post-translational modifications of one or more proteins.

As used herein, the terms “dysregulation” and “dysregulated” refer to situations or conditions wherein homeostatic control systems have been disturbed and/or compromised so that one or more metabolic, physiologic and/or biochemical systems within a subject operate partially or entirely without said homeostatic control systems.

As used herein, the term “endogenous” refers to the production and/or modification of a molecule that originates within a subject.

As used herein, the term “exogenous” refers to a molecule that is within a subject but that did not originate within the subject.

As used herein, the terms “inhibit”, “inhibiting”, and “inhibition” refer to a decrease in activity, response, or other biological parameter of a biologic process, disease, disorder or symptom thereof. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between the specifically recited percentages, as compared to native or control levels.

As used herein, the phrases “prevention of” and “preventing” refer to avoiding the onset or progression of a disease, disorder, or a symptom thereof.

As used herein, the terms “production”, “producing” and “produce” refer to the synthesis and/or replication of DNA, the transcription of one or more sequences of RNA, the translation of one or more amino acid sequences, the post-translational modifications of an amino acid sequence, and/or the production of one or more regulatory molecules that can influence the production and/or functionality of an effector molecule or an effector cell. For clarity, “production” is also be used herein to refer to the functionality of a regulatory molecule, unless the context reasonably indicates otherwise.

As used herein, the terms “promote”, “promotion”, and “promoting” refer to an increase in an activity, response, condition, disease process, or other biological parameter. This can include, but is not limited to, the initiation of the activity, response, condition, or disease process. This may also include, for example, a 10% increase in the activity, response, condition, or disease as compared to the native or control level. Thus, the increase in an activity, response, condition, disease, or other biological parameter can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, including any amount of increase in between the specifically recited percentages, as compared to native or control levels.

As used herein, the term “subject” refers to any therapeutic target that receives the agent. The subject can be a vertebrate, for example, a mammal including a human. The term “subject” does not denote a particular age or sex. The term “subject” also refers to one or more cells of an organism, an in vitro culture of one or more tissue types, an in vitro culture of one or more cell types, ex vivo preparations, and/or a sample of biological materials such as tissue and/or biological fluids.

As used herein, the term “target cell” refers to one or more cells and/or cell types that are deleteriously affected, either directly or indirectly, by a dysregulated immune system and/or a disease process. The term “target cell” also refers to cells that are not deleteriously affected but that are the cells in which it is desired that the agent interacts.

As used herein, the term “therapeutically effective amount” refers to the amount of the agent used that is of sufficient quantity to ameliorate, treat and/or inhibit one or more of a disease, disorder or a symptom thereof. The “therapeutically effective amount” will vary depending on the agent used, the route of administration of the agent and the severity of the disease, disorder or symptom thereof. The subject's age, weight and genetic make-up may also influence the amount of the agent that will be a therapeutically effective amount.

As used herein, the terms “treat”, “treatment” and “treating” refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing an occurrence of a disease, disorder or symptom thereof and/or the effect may be therapeutic in providing a partial or complete amelioration or inhibition of a disease, disorder, or symptom thereof. Additionally, the term “treatment” refers to any treatment of a disease, disorder, or symptom thereof in a subject and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) ameliorating the disease.

As used herein, the terms “unit dosage form” and “unit dose” refer to a physically discrete unit that is suitable as a unitary dose for patients. Each unit contains a predetermined quantity of the agent and optionally, one or more suitable pharmaceutically acceptable carriers, one or more excipients, one or more additional active ingredients, or combinations thereof. The amount of agent within each unit is a therapeutically effective amount.

Where a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also, encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Some embodiments of the present disclosure relate to a recombinant plasmid (RP) for introducing genetic material, such as one or more nucleotide sequences, into a target cell for reproduction or transcription of an insert that comprises one or more nucleotide sequences that are carried within the RP. In some embodiments of the present disclosure, the RP is delivered by a viral vector. In some embodiments of the present disclosure, the vector is an adeno-associated virus vector.

In some embodiments of the present disclosure, the insert comprises one or more nucleotide sequences that encode for production of at least a belatacept similar protein (BSP).

The BSP has physiologic/biologic equivalence to belatacept, meaning the BSP will have substantially the same effect on the subject or target cell as a similar dose of belatacept will. Belatacept is a known immunosuppressant that is used to reduce rejection in recipients of organ transplants. Similar to belatacept, BSP is a fusion protein that combines an Fc portion of a human immunoglobulin IgG1 with an extracellular portion of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Belatacept can interfere with binding of antigen presenting cells and, therefore, belatacept can prevent activation of T-cells. As such, the BSP may also prevent activation of T-cells.

In some embodiments of the present disclosure, the insert comprises one or more nucleotide sequences that encode for production of at least one sequence of miRNA that decreases the production of target cytokine proteins. The miRNA may, directly or indirectly, bind to and degrade the target mRNA or otherwise inactivate the target mRNA so that less or none of the target-cytokine protein is produced.

In some embodiments of the present disclosure, the target cytokine is a pro-inflammatory cytokine, meaning it has the physiologic effect of increasing inflammatory processes in the subject. In some embodiments of the present disclosure, the target cytokine is TNF-alpha. In some embodiments of the present disclosure, the target cytokine is an anti-inflammatory cytokine.

In some embodiments of the present disclosure, the insert comprises two or more nucleotide sequences that each encode one or more miRNA sequences that may be complimentary to and degrade, or cause degradation of, mRNA of the target cytokine. In some embodiments of the present disclosure the insert comprises three nucleotide sequences that each encode an miRNA sequence that may be complimentary to and degrade, or causes degradation of, or inactivates or causes inactivation of mRNA of the target cytokine.

In some embodiments of the present disclosure, the insert comprises one or more nucleotide sequences that encode for production of the BSP and one or more nucleotide sequences that each encode for an miRNA sequence that is complimentary to and degrades, or causes degradation of, or inactivates or causes inactivation of mRNA of the target cytokine. In some embodiments of the present disclosure, the insert comprises two nucleotide sequences that each encode for miRNA that may be complimentary to and degrade, or causes degradation of, or inactivate, or causes inactivation of mRNA of the target cytokine. In some embodiments of the present disclosure, the insert comprises three nucleotide sequences that each encode for miRNA that may be complimentary to and degrade, or causes degradation of, or inactivate, or causes inactivation of mRNA of the target cytokine. In some embodiments of the present disclosure, the insert comprises more than three nucleotide sequences that each encode for miRNA that may be complimentary to and degrade, or causes degradation of, or inactivate, or causes inactivation of mRNA of the target cytokine.

The present disclosure relates to one or more agents, therapies, treatments, and methods of use of the agents and/or therapies and/or treatments for initiating or upregulating production of the BSP while downregulating production and/or functionality of the target cytokine. Some embodiments of the present disclosure relate to methods for making a complex between at least one particle of an agent and at least one target cell of a subject for initiating or increasing production of the BSP and for downregulating the subject's production and/or functionality of the target cytokine. Embodiments of the present disclosure can be used as a therapy or a treatment for a subject that has a condition whereby the subject's immune system is, or is likely to become, dysregulated.

In some embodiments of the present disclosure, the agent can be administered to the subject by an intravenous route, an intramuscular route, an intraperitoneal route, an intrathecal route, an intravesical route, a topical route, an intranasal route, a transmucosal route, a pulmonary route, and combinations thereof.

In some embodiments of the present disclosure, the agent can be administered to the subject by pipetting a dose of the agent into an in vitro cell culture, perfusing or immersing an ex vivo cell or tissue preparation with a solution that comprises the agent, mixing a biological fluid sample with a solution or substrate that comprises the agent, or combinations thereof.

Some embodiments of the present disclosure relate to an agent that can be administered to a subject with a condition that results from, directly or indirectly, a dysregulated immune system. When a therapeutically effective amount of the agent is administered to the subject, the subject may change production and/or functionality of one or more immune system molecules. For example, the subject may decrease production and/or functionality of one or more immune system signaling molecules and/or one or more immune system effector molecules by changing the production of one or more sequences of DNA, one or more sequences of RNA and/or one or more proteins and/or one or more regulatory molecules that regulate the levels and/or functionality of the subject's immune system signaling molecules and/or immune system effector molecules.

In some embodiments of the present disclosure, the subject may respond to receiving the therapeutic amount of the agent by changing production and/or functionality of one or more intermediary molecules by changing production of one or more DNA sequences, one or more RNA sequences, and/or one or more proteins that regulate the levels and/or functionality of the one or more intermediary molecules. The one or more intermediary molecules regulate the subject's levels and/or functionality of the one or more immune system signaling molecules and/or the one or more immune system effector molecules.

In some embodiments of the present disclosure, administering a therapeutic amount of the agent to a subject upregulates the production, functionality or both of the BSP and one or more sequences of miRNA that each target the mRNA of one or more target cytokines. Examples of the target cytokine include one or more pro-inflammatory cytokines, one or more anti-inflammatory cytokines or combinations thereof. In some embodiments of the present disclosure, there are one, two three, four, five, six, seven, eight, nine or ten miRNA sequences that each are complimentary to and degrade, or cause degradation of, one cytokine, such as TNF-alpha. In some embodiments of the present disclosure, the agent may comprise multiple copies of the same nucleotide sequence of miRNA.

In some embodiments of the present disclosure, the composition is an RP that may be used for gene therapy. The gene therapy is useful for increasing the subject's endogenous production of the BSP and one or more sequences of miRNA that target the mRNA of a target cytokine. For example, the RP can contain one or more nucleotide sequences that that cause increased production of the BSP and one or more nucleotide sequences that cause an increased production of one or more miRNA sequences that that each are complimentary to and degrade, or cause degradation of, or inactivate, or cause inactivation of one cytokine, such as TNF-alpha.

In some embodiments of the present disclosure, the RP can be carried by a vector—also referred to as a delivery vehicle—that is a virus used for gene therapy that can be enveloped or not (undeveloped), replication effective or not (replicative ineffective), or combinations thereof. In some embodiments of the present disclosure, the vector is a virus that is not enveloped and replication ineffective. In some embodiments of the present disclosure, the vector is a virus of the Paroviridae family. In some embodiments of the present disclosure, the vector is a virus of the genus Dependoparvaovirus. In some embodiments of the present disclosure, the vector is an adeno-associated virus (AAV). In some embodiments of the present disclosure, the vector is a recombinant AAV. In some embodiments of the present disclosure, the vector is a recombinant AAV6.2FF.

The embodiments of the present disclosure also relate to administering a therapeutically effective amount of the agent. In some embodiments of the present disclosure, the therapeutically effective amount of the agent that is administered to a patient is between about 10 and about 1×10¹⁶TCID₅₀/kg (50% tissue culture infective dose per kilogram of the patient's body weight). In some embodiments of the present disclosure, the therapeutically effective amount of the agent that is administered to the patient is about 1×10¹³TCID₅₀/kg. In some embodiments of the present disclosure, the therapeutically effective amount of the agent that is administered to a patient is measured in TPC/kg (total particle count of the agent per kilogram of the patient's body weight). In some embodiments the therapeutically effective amount of the agent is between about 10 and about 1×10¹⁶TCP/kg.

Some embodiments of the present disclosure relate to an adenovirus associated virus (AAV) genome comprising an RP that when operable inside a target cell will cause the target cell to produce both the BSP and a miRNA sequence that downregulates production of TNF-α. The AAV vector is comprised of AAV2 inverted terminal repeats (ITRs), a composite CASI promoter, the coding sequence for the BSP with a human growth hormone (HGH) signal peptide followed by a miR155 microRNA cassette containing three different siRNAs targeting murine TNF-α, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and an SV40 polyA signal.

SEQ ID No. 1 (whole RP):

cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
120

atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
180

tgctctagga cattgattat tgactagtgg agttccgcgt tacataactt acggtaaatg
240

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
300

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
360

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
420

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
480

cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
540

tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
600

tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggcgcgcgc caggcggggc
660

ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
720

cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
780

gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
840

cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
900

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
960

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
1020

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
1080

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
1140

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
1200

gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
1260

cagggtaccg ccaccatggc caccggctct cgcacaagcc tgctgctggc tttcggactg
1320

ctgtgcctgc cttggctcca ggagggctcc gccatgcacg tggcccagcc tgctgtggta
1380

ctggccagca gccgaggcat cgccagcttt gtgtgtgagt atgcatctcc aggcaaatac
1440

actgaggtcc gggtgacagt gcttcggcag gctgacagcc aggtgactga agtctgtgcg
1500

gcaacctaca tgatggggaa tgagttgacc ttcctagatg attccatctg cacgggcacc
1560

tccagtggaa atcaagtgaa cctcactatc caaggactga gggccatgga cacgggactc
1620

tacatctgca aggtggagct catgtaccca ccgccatact acgagggcat aggcaacgga
1680

acccagattt atgtaattga tccagaaccg tgcccagatt ctgatcaggg atccggagga
1740

ggaggcagcg gaggaggcgg atctggcgga ggcggaagcg acaagaccca cacatgccca
1800

ccatgtcctg cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc
1860

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc
1920

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
1980

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
2040

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc
2100

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag
2160

gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc
2220

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
2280

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
2340

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg
2400

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa
2460

taggctagca tcgataccgt cgctatgtgc tggaggcttg ctgaaggctg tatgctggct
2520

cctccacttg gtggtttggt tttggcctct gactgaccaa accacagtgg aggagccagg
2580

acacaaggcc tgttactagc actcacatgg aacaaatggc ctctagcctg gaggcttgct
2640

gaaggctgta tgctgtttct cctggtatga gatagcgttt tggcctctga ctgacgctat
2700

ctcaccagga gaaacaggac acaaggcctg ttactagcac tcacatggaa caaatggcct
2760

ctagcctgga ggcttgctga aggctgtatg ctgccgcaaa gtctaagtac ttgggttttg
2820

gcctctgact gacccaagta cagactttgc ggcaggacac aaggcctgtt actagcactc
2880

acatggaaca aatggcctct ctagaataat caacctctgg attacaaaat ttgtgaaaga
2940

ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
3000

cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
3060

tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
3120

actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
3180

tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
3240

gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
3300

aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
3360

tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
3420

ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
3480

tgggccgcct ccccgcctaa gottatcgat accgtcgaga tctaacttgt ttattgcagc
3540

ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
3600

actgcattct agttgtggtt tgtccaaact catcaatgta tottatcatg tctggatctc
3660

gacctcgact agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca
3720

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
3780

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
3840

gagcgcgcag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
3900

gcagcctgaa tggcgaatgg cgattccgtt gcaatggctg gcggtaatat tgttctggat
3960

attaccagca aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat
4020

caaagaagta ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc
4080

ctcactgatt ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct
4140

ttaatcggcc tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg
4200

ctcgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
4260

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
4320

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
4380

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
4440

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
4500

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
4560

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
4620

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttaaat
4680

atttgcttat acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat
4740

gattgacatg ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc
4800

aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg
4860

aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt
4920

tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag
4980

ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag
5040

ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat
5100

tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaattcc tgatgcggta
5160

ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
5220

ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
5280

ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
5340

ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
5400

gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
5460

cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
5520

tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
5580

gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
5640

tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
5700

tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
5760

ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
5820

atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
5880

cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
5940

attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
6000

gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
6060

ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
6120

gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
6180

agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
6240

gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
6300

gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
6360

ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
6420

tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
6480

tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
6540

catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
6600

gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
6660

aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
6720

gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
6780

gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
6840

gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
6900

atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
6960

cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
7020

cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
7080

agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
7140

tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
7200

gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
7260

catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
7320

agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
7380

ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatg
7437

(nucleotide sequence for BSP)

SEQ ID No. 2

atgcacgtgg cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg
60

tgtgagtatg catctccagg caaatacact gaggtccggg tgacagtgct tcggcaggct
120

gacagccagg tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc
180

ctagatgatt ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa
240

ggactgaggg ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg
300

ccatactacg agggcatagg caacggaacc cagatttatg taattgatcc agaaccgtgc
360

ccagattctg atcag
375

(miRNA cassette)

SEQ ID NO. 3

ctggaggctt gctgaaggct gtatgctggc tcctccactt ggtggtttgg ttttggcctc
60

tgactgacca aaccacagtg gaggagccag gacacaaggc ctgttactag cactcacatg
120

gaacaaatgg cctctagcct ggaggcttgc tgaaggctgt atgctgtttc tcctggtatg
180

agatagcgtt ttggcctctg actgacgcta tctcaccagg agaaacagga cacaaggcct
240

gttactagca ctcacatgga acaaatggcc tctagcctgg aggcttgctg aaggctgtat
300

gctgccgcaa agtctaagta cttgggtttt ggcctctgac tgacccaagt acagactttg
360

cggcaggaca caaggcctgt tactagcact cacatggaac aaatggcctc tctagaa
417

(nucleotide sequence for 5′ miRNA-155 #1-3′miRNA#1-155)

SEQ ID No. 4

ctggaggctt gctgaaggct gtatgctggc tcctccactt ggtggtttgg ttttggcctc
60

tgactgacca aaccacagtg gaggagccag gacacaaggc ctgttactag cactcacatg
120

gaacaaatgg cc
132

(nucleotide sequence for 5′ miRNA-155 #2-3′miRNA #2)

SEQ ID No. 5

ctggaggctt gctgaaggct gtatgctgtt tctcctggta tgagatagcg ttttggcctc
60

tgactgacgc tatctcacca ggagaaacag gacacaaggc ctgttactag cactcacatg
120

gaacaaatgg cc
132

(nucleotide sequence for 5′ miRNA-155 #3-3′ miRNA #3)

SEQ ID No. 6

ctggaggctt gctgaaggct gtatgctgcc gcaaagtcta agtacttggg ttttggcctc
60

tgactgaccc aagtacagac tttgcggcag gacacaaggc ctgttactag cactcacatg
120

gaacaaatgg cc
132

(miRNA 1 anti-sense)

SEQ ID No. 7

gctcctccac ttggtggttt g
21

(miRNA 1 sense)

SEQ ID No. 8

caaaccacag tggaggagc
19

(miRNA 2 anti-sense)

SEQ ID No. 9

tttctcctgg tatgagatag c
21

(miRNA 2 sense)

SEQ ID No. 10

gctatctcac caggagaaa
19

(miRNA 3 anti-sense)

SEQ ID No. 11

ccgcaaagtc taagtacttg g
21

(miRNA 3 sense)

SEQ ID No. 12

ccaagtacag actttgcgg
19

(nucleotide sequence for Start of whole RP to start of BSP)

SEQ ID No. 13

cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
120

atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
180

tgctctagga cattgattat tgactagtgg agttccgcgt tacataactt acggtaaatg
240

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
300

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
360

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
420

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
480

cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
540

tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
600

tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggcgcgcgc caggcggggc
660

ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
720

cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
780

gcgaagcgcg cggCgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
840

cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
900

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
960

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
1020

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
1080

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
1140

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
1200

gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
1260

cagggtaccg ccaccatggc caccggctct cgcacaagcc tgctgctggc tttcggactg
1320

ctgtgcctgc cttggctcca ggagggctcc gcc
1353

(nucleotide sequence for Start of whole RP to end of 3′ miRNA #1)

SEQ ID No. 14

cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
120

atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
180

tgctctagga cattgattat tgactagtgg agttccgcgt tacataactt acggtaaatg
240

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
300

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
360

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
420

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
480

cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
540

tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
600

tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggcgcgcgc caggcggggc
660

ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
720

cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggCggc cctataaaaa
780

gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
840

cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
900

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
960

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
1020

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
1080

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
1140

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
1200

gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
1260

cagggtaccg ccaccatggc caccggctct cgcacaagcc tgctgctggc tttcggactg
1320

ctgtgcctgc cttggctcca ggagggctcc gccatgcacg tggcccagcc tgctgtggta
1380

ctggccagca gccgaggcat cgccagcttt gtgtgtgagt atgcatctcc aggcaaatac
1440

actgaggtcc gggtgacagt gcttcggcag gctgacagcc aggtgactga agtctgtgcg
1500

gcaacctaca tgatggggaa tgagttgacc ttcctagatg attccatctg cacgggcacc
1560

tccagtggaa atcaagtgaa cctcactatc caaggactga gggccatgga cacgggactc
1620

tacatctgca aggtggagct catgtaccca ccgccatact acgagggcat aggcaacgga
1680

acccagattt atgtaattga tccagaaccg tgcccagatt ctgatcaggg atccggagga
1740

ggaggcagcg gaggaggcgg atctggcgga ggcggaagcg acaagaccca cacatgccca
1800

ccatgtcctg cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc
1860

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc
1920

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
1980

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
2040

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc
2100

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag
2160

gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc
2220

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
2280

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
2340

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg
2400

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa
2460

taggctagca tcgataccgt cgctatgtgc tggaggcttg ctgaaggctg tatgctggct
2520

cctccacttg gtggtttggt tttggcctct gactgaccaa accacagtgg aggagccagg
2580

acacaaggcc tgttactagc actcacatgg aacaaatggc c
2621

(nucleotide sequence for Start of whole RP to end of 3′ miRNA #2)

SEQ ID No. 15

cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
120

atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
180

tgctctagga cattgattat tgactagtgg agttccgcgt tacataactt acggtaaatg
240

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
300

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
360

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
420

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
480

cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
540

tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
600

tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggcgcgcgc caggcggggc
660

ggggcggggc gaggggCggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
720

cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
780

gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
840

cgccgcctcg cgccgcccGc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
900

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
960

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
1020

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
1080

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
1140

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
1200

gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
1260

cagggtaccg ccaccatggc caccggctct cgcacaagcc tgctgctggc tttcggactg
1320

ctgtgcctgc cttggctcca ggagggctcc gccatgcacg tggcccagcc tgctgtggta
1380

ctggccagca gccgaggcat cgccagcttt gtgtgtgagt atgcatctcc aggcaaatac
1440

actgaggtcc gggtgacagt gcttcggcag gctgacagcc aggtgactga agtctgtgcg
1500

gcaacctaca tgatggggaa tgagttgacc ttcctagatg attccatctg cacgggcacc
1560

tccagtggaa atcaagtgaa cctcactatc caaggactga gggccatgga cacgggactc
1620

tacatctgca aggtggagct catgtaccca ccgccatact acgagggcat aggcaacgga
1680

acccagattt atgtaattga tccagaaccg tgcccagatt ctgatcaggg atccggagga
1740

ggaggcagcg gaggaggcgg atctggcgga ggcggaagcg acaagaccca cacatgccca
1800

ccatgtcctg cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc
1860

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc
1920

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
1980

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
2040

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc
2100

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag
2160

gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc
2220

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
2280

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
2340

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg
2400

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa
2460

taggctagca tcgataccgt cgctatgtgc tggaggcttg ctgaaggctg tatgctggct
2520

cctccacttg gtggtttggt tttggcctct gactgaccaa accacagtgg aggagccagg
2580

acacaaggcc tgttactagc actcacatgg aacaaatggc ctctagcctg gaggcttgct
2640

gaaggctgta tgctgtttct cctggtatga gatagcgttt tggcctctga ctgacgctat
2700

ctcaccagga gaaacaggac acaaggcctg ttactagcac tcacatggaa caaatggcc
2759

(nucleotide sequence for Start of whole RP to end of 3′ miRNA #3)

SEQ ID No. 16

cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
120

atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
180

tgctctagga cattgattat tgactagtgg agttccgcgt tacataactt acggtaaatg
240

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
300

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
360

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
420

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
480

cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
540

tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
600

tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggcgcgcgc caggcggggc
660

ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
720

cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
780

gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
840

cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
900

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
960

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
1020

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
1080

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
1140

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
1200

gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
1260

cagggtaccg ccaccatggc caccggctct cgcacaagcc tgctgctggc tttcggactg
1320

ctgtgcctgc cttggctcca ggagggctcc gccatgcacg tggcccagcc tgctgtggta
1380

ctggccagca gccgaggcat cgccagcttt gtgtgtgagt atgcatctcc aggcaaatac
1440

actgaggtcc gggtgacagt gcttcggcag gctgacagcc aggtgactga agtctgtgcg
1500

gcaacctaca tgatggggaa tgagttgacc ttcctagatg attccatctg cacgggcacc
1560

tccagtggaa atcaagtgaa cctcactatc caaggactga gggccatgga cacgggactc
1620

tacatctgca aggtggagct catgtaccca ccgccatact acgagggcat aggcaacgga
1680

acccagattt atgtaattga tccagaaccg tgcccagatt ctgatcaggg atccggagga
1740

ggaggcagcg gaggaggcgg atctggcgga ggcggaagcg acaagaccca cacatgccca
1800

ccatgtcctg cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc
1860

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc
1920

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
1980

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
2040

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc
2100

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag
2160

gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc
2220

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
2280

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
2340

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg
2400

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa
2460

taggctagca tcgataccgt cgctatgtgc tggaggcttg ctgaaggctg tatgctggct
2520

cctccacttg gtggtttggt tttggcctct gactgaccaa accacagtgg aggagccagg
2580

acacaaggcc tgttactagc actcacatgg aacaaatggc ctctagcctg gaggcttgct
2640

gaaggctgta tgctgtttct cctggtatga gatagcgttt tggcctctga ctgacgctat
2700

ctcaccagga gaaacaggac acaaggcctg ttactagcac tcacatggaa caaatggcct
2760

ctagcctgga ggcttgctga aggctgtatg ctgccgcaaa gtctaagtac ttgggttttg
2820

gcctctgact gacccaagta cagactttgc ggcaggacac aaggcctgtt actagcactc
2880

acatggaaca aatggcc
2897

(inverted terminal repeat #1)

SEQ ID No. 17

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
120

aggggttcct
130

(ITR #1 to ITR #2)

SEQ ID No. 18

cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
120

atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
180

tgctctagga cattgattat tgactagtgg agttccgcgt tacataactt acggtaaatg
240

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
300

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
360

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
420

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
480

cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
540

tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
600

tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggcgcgcgc caggcggggc
660

ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
720

cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
780

gcgaagcgcg cggCgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
840

cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
900

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
960

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
1020

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
1080

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
1140

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
1200

gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
1260

cagggtaccg ccaccatggc caccggctct cgcacaagcc tgctgctggc tttcggactg
1320

ctgtgcctgc cttggctcca ggagggctcc gccatgcacg tggcccagcc tgctgtggta
1380

ctggccagca gccgaggcat cgccagcttt gtgtgtgagt atgcatctcc aggcaaatac
1440

actgaggtcc gggtgacagt gcttcggcag gctgacagcc aggtgactga agtctgtgcg
1500

gcaacctaca tgatggggaa tgagttgacc ttcctagatg attccatctg cacgggcacc
1560

tccagtggaa atcaagtgaa cctcactatc caaggactga gggccatgga cacgggactc
1620

tacatctgca aggtggagct catgtaccca ccgccatact acgagggcat aggcaacgga
1680

acccagattt atgtaattga tccagaaccg tgcccagatt ctgatcaggg atccggagga
1740

ggaggcagcg gaggaggcgg atctggcgga ggcggaagcg acaagaccca cacatgccca
1800

ccatgtcctg cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc
1860

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc
1920

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
1980

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
2040

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc
2100

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag
2160

gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc
2220

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
2280

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
2340

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg
2400

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa
2460

taggctagca tcgataccgt cgctatgtgc tggaggcttg ctgaaggctg tatgctggct
2520

cctccacttg gtggtttggt tttggcctct gactgaccaa accacagtgg aggagccagg
2580

acacaaggcc tgttactagc actcacatgg aacaaatggc ctctagcctg gaggcttgct
2640

gaaggctgta tgctgtttct cctggtatga gatagcgttt tggcctctga ctgacgctat
2700

ctcaccagga gaaacaggac acaaggcctg ttactagcac tcacatggaa caaatggcct
2760

ctagcctgga ggcttgctga aggctgtatg ctgccgcaaa gtctaagtac ttgggttttg
2820

gcctctgact gacccaagta cagactttgc ggcaggacac aaggcctgtt actagcactc
2880

acatggaaca aatggcctct ctagaataat caacctctgg attacaaaat ttgtgaaaga
2940

ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
3000

cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
3060

tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
3120

actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
3180

tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
3240

gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
3300

aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
3360

tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
3420

ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
3480

tgggccgcct ccccgcctaa gcttatcgat accgtcgaga tctaacttgt ttattgcagc
3540

ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
3600

actgcattct agttgtggtt tgtccaaact catcaatgta tottatcatg tctggatctc
3660

gacctcgact agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca
3720

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
3780

ccgggcgacc aaaggtcgcc cgacgcccgg getttgcccg ggcggcctca gtgagcgagc
3840

gagcgcgcag
3850

(inverted terminal repeat #2)

SEQ ID No. 19

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
120

gagcgcgcag
130

(amino acid sequence for BSP produced per SEQ ID No. 2)

SEQ ID No. 20

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile

100 105 110

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

115 120 125

Example 1—Expression Cassette

Expression cassettes for expressing a monoclonal antibody (mAb) and/or a protein and/or miRNA were synthesized by Genscript. Each cassette contained a signal peptide, the variable heavy domain, the human IgG1 constant domain, the protein or the miRNA sequence followed by (when it is an Ab), a self-cleaving 2A peptide sequence, a signal peptide, the variable light domain and the human lambda constant domain. The synthesized mAb and/or protein and/or miRNA expression cassettes were cloned into the pAVA-00200 plasmid backbone containing the CASI promoter1, multiple cloning site (MCS), Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), Simian virus 40 (SV40) polyadenylation (polyA) sequence all flanked by the AAV2 inverted terminal repeats (ITR). pAVA-00200 was cut with the restriction enzymes KpnI and XbaI in the MCS and separated on a 1% agarose gel. The band of interest was excised and purified using a gel extraction kit. Each mAb and/or protein and/or miRNA expression cassette was amplified by PCR using Taq polymerase and the PCR products were gel purified and the bands on interest were also excised and purified using a gel extraction kit. These PCR products contained the mAb and/or protein and/or miRNA expression cassettes in addition to 15 base pair 5′ and 3′ overhangs that align with the ends of the linearized pAVA-00200 backbone. Using in-fusion cloning2, the amplified mAb or protein or miRNA expression cassettes are integrated with the pAVA-00200 backbone via homologous recombination. The resulting plasmid vectors contained the following 5′ ITR, CASI promoter, monoclonal antibody or protein or miRNA expression cassette, WPRE, SV40 polyA and ITR 3′, per SEQ ID No. 1 and as shown in FIG. 1 and as shown in fourteen contiguous portions in FIG. 2 through FIG. 15.

Example 2—Animal Studies

C57BL/6 mice and BALB/c mice were purchased from Charles River. AAV vectors of Example 1 were administered to 6-week-old C57BL/6 mice with the exception of the AAV vector that encoded mAb expression, which was tested in BALB/c mice. All animal experiments were approved by the institutional animal care committees of the Canadian Science Centre for Human and Animal Health and the University of Guelph. Intramuscular or intraorgan administration of the AAV were performed using a 29-gauge needle and a 40-μL injection volume. Injection into the tail vein was performed on mice that were warmed slightly, using a 100-μL injection volume. Intranasal administration of the AAV vectors were performed using a 40-μL injection volume. The dose used was about 2×10¹¹vector genomes per mouse.

Example 3—Experimental Data

FIG. 17 is an image of a Western blot gel that shows BSP expression in samples taken from muscle of mice administered i.m. (gastrocnemius muscle) 1×10¹¹vg of the composition of SEQ ID No. 1. The samples were taken 28 days post-administration. Lysates were probed with an anti-hIgG1 antibody. Lane 1 is a molecular weight marker, lane 2 is a mock sample, lane 3 is a further mock sample, lanes 4 and 5 are samples taken from mice administered the composition with SEQ ID No. 1. The BSP proteins can be seen in as a gel migrant with an approximate molecular weight of 48 kDa in lanes 4 and 5, whereas samples obtains from mock administered mice (lanes 2 and 3) did not show any gel migratory bands at 48 kDa. Without being bound by any particular theory, administering the composition induced mice to produce BSP.

FIG. 18 shows a scatter plot of human IgG (ng/4) measured in plasma samples obtained at day 0, 7, 14, 21 and 28 post-administration of the composition with of SEQ ID No. 1. The plasma samples were analyzed using a commercially available human IgG quantitative ELISA (Abcam cat no. 195215). Without being bound by any particular theory, administering the composition induced mice to produce human IgG 1.

Example 4—In Vitro Studies

FIG. 19 is an image of another Western blot gel that shows a reduction in recombinant TNF-α expression in HEK293 cells transduced with the RP of SEQ ID NO. 1, 5 days prior to transfection with a mammalian expression vector encoding murine TNF-α fused to a green fluorescence protein (GFP) and harvested 3 days later. In order to obtain samples for protein analysis, lysates were probed with an anti-murine TNF-α antibody. In the image of the gel, lanes 1 and 5 each contained mock samples, lanes 2 and 6 each contained a sample taken from GFP-TNF-α transfected cells, lanes 3 and 7 contain samples from cells transfected with the composition of SEQ ID No. 1 and lanes 4 and 8 contain samples from cells transfected with the vector of SEQ ID No. 1 and the GFP-TNF-α. The bottom portion of the gel image shows gel migratory bands that correspond with an anti-GAPDH antibody, demonstrating substantially equal loading of total protein in the gel lanes. The samples with GFP-TNF-α show a gel migrant with an approximate molecular weight of 54 kDa. Without being bound by any particular theory, these results demonstrate efficacy of miRNA-based gene-silencing of TNF-α production. The engineered miRNAs produced by the miR155 fully complement their target site and cleave the target mRNA, in this case TNF-α leading to a reduction in TNF-α protein expression.

Table 1 below shows the expression levels of TNF-alpha (ag) measured in HEK293 cells transduced with an AAV that included a nucleotide sequence that encoded for TNF-alpha siRNA, TNF-alpha miRNA or the BSP and TNF-alpha miRNA (SEQ ID No. 1) or a respective control.

TABLE 1

TNF-Alpha expression following administration

of three AAVs.

Control
AAV

(ag)
recipient (ag)
P-value

TNF-alpha siRNA
12
2
0.0151

TNF-alpha miRNA
2273
130
<0.001

TNF-alpha miRNA/BSP
2311
142
<0.001

COMPOSITIONS AND METHODS FOR REGULATING PRODUCTION OF A FUSION PROTEIN AND RIBONUCLEIC ACID

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